Modulation of glucagon receptor expression

ABSTRACT

Compounds, compositions and methods are provided for modulating the expression of glucagon receptor. The compositions comprise oligonucleotides, targeted to nucleic acid encoding glucagon receptor. Methods of using these compounds for modulation of glucagon receptor expression and for diagnosis and treatment of disease associated with expression of glucagon receptor are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/697,280, filed Apr. 5, 2007, which is a divisional of U.S.application Ser. No. 10/832,777, filed Apr. 27, 2004, now U.S. Pat. No.7,399,853, issued Jul. 15, 2008, which claims benefit of U.S.provisional application 60/466,256, filed Apr. 28, 2003, the entirecontents of each is being expressly incorporated herein by reference.

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledBIOL0007USC1SEQ.txt, created on Feb. 9, 2010 which is 208 Kb in size.The information in the electronic format of the sequence listing isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention provides compositions and methods for modulatingthe expression of glucagon receptor. In particular, this inventionrelates to compounds, particularly oligonucleotide compounds, which, inpreferred embodiments, hybridize with nucleic acid molecules encodingglucagon receptor. Such compounds are shown herein to modulate theexpression of glucagon receptor.

BACKGROUND OF THE INVENTION

The maintenance of normal glycemia is a carefully regulated metabolicevent. Glucagon, the 29-amino acid peptide responsible for maintainingblood glucose levels in the postabsorbative state, increases glucoserelease from the liver by activating hepatic glycogenolysis,gluconeogenesis, stimulating lipolysis in adipose tissue, andstimulating insulin secretion. During high blood glucose levels, insulinreverses the glucagon-mediated enhancement of glycogenolysis andgluconeogenesis. In patients with diabetes, insulin is either notavailable or not fully effective. While treatment for diabetes hastraditionally focused on increasing insulin levels, antagonism ofglucagon function has been considered as an alternative therapy. Asglucagon exerts its physiological effects by signaling through theglucagon receptor, the glucagon receptor has been proposed as apotential therapeutic target for diabetes (Madsen et al., Curr. Pharm.Des., 1999, 5, 683-691).

Glucagon receptor is belongs to the superfamily of G-protein-coupledreceptors having seven transmembrane domains. It is also a member of thesmaller sub-family of homologous receptors which bind peptides that arestructurally similar to glucagon. The gene encoding human glucagonreceptor was cloned in 1994 and analysis of the genomic sequencerevealed multiple introns and an 82% identity to the rat glucagonreceptor gene (Lok et al., Gene, 1994, 140, 203-209.; MacNeil et al.,Biochem. Biophys. Res. Commun., 1994, 198, 328-334). Cloning of the ratglucagon receptor gene also led to the description of multiplealternative splice variants (Maget et al., FEBS Lett., 1994, 351,271-275). Disclosed and claimed in U.S. Pat. No. 5,776,725 is anisolated nucleic acid sequence encoding a human or rat glucagon receptor(Kindsvogel et al., 1998). The human glucagon receptor gene is localizedto chromosome 17q25 (Menzel et al., Genomics, 1994, 20, 327-328). Amissense mutation of Gly to Ser at codon 40 in the glucagon receptorgene leads to a 3-fold lower affinity for glucagon (Fujisawa et al.,Diabetologia, 1995, 38, 983-985) and this mutation has been linked toseveral disease states, including non-insulin-dependent diabetesmellitus (Fujisawa et al., Diabetologia, 1995, 38, 983-985),hypertension (Chambers and Morris, Nat. Genet., 1996, 12, 122), andcentral adiposity (Siani et al., Obes. Res., 2001, 9, 722-726).

Inhibiting glucagon function by antagonizing the glucagon receptor hasbeen proposed as a therapeutic target for diabetes. Currently, there areno known therapeutic agents which effectively inhibit the synthesis ofglucagon receptor and to date, investigative strategies aimed atmodulating glucagon receptor function have involved the use ofantibodies, peptidyl antagonists, and small molecules. In addition,targeted disruption of the glucagon receptor gene in mice has shownthat, despite a total absence of glucagon receptors and elevated plasmaglucagon levels, the mice maintain near-normal glycemia and lipidemia(Parker et al., Biochem. Biophys. Res. Commun., 2002, 290, 839-843).Patent application WO 02/45494 (Allen et al.) discloses transgenic micecomprising mutations in a glucagon receptor gene. Also claimed areagonists or antagonists of glucagon receptor, agents that modulate thefunction, expression or activity of a glucagon receptor gene, methods ofidentifying such agents, methods of ameliorating conditions associatedwith impaired glucose tolerance, methods of identifying agents thataffect obesity, weight gain, diabetes, methods of treating obesity ordiabetic conditions, and phenotypic data associated with a transgenicmouse comprising a mutation in a glucagon receptor gene.

A glucagon-neutralizing monoclonal antibody has been described thatantagonizes glucagon-stimulated signal transduction in part by bindingto the glucagon binding site of the glucagon receptor (Buggy et al.,Horm. Metab. Res., 1996, 28, 215-219). An antibody which specificallybinds to the amino acid sequence of a glucagon receptor has beendisclosed and claimed in U.S. Pat. No. 5,770,445 (Kindsvogel et al.,1998).

Several peptidyl antagonists of glucagon receptor have been reported inthe art. Six glucagon analogs with N-terminal modifications weredesigned to have a higher affinity than glucagon for the glucagonreceptor (Zechel et al., Int. J. Pept. Protein Res., 1991, 38, 131-138).Two somatostatin analogs have been reported to be inhibitors of glucagonsecretion (Rossowski and Coy, Biochem. Biophys. Res. Commun., 1994, 205,341-346).

Many small molecules have been examined as glucagon receptor antagonistsincluding: [(+)-3,5diisopropyl-2-(1-hydroxyethyl)-6-propyl-4′-fluoro-1,1′-biphenyl(Bay27-9955) (Petersen and Sullivan, Diabetologia, 2001, 44, 2018-2024),a series of alkylidene hydrazides (Ling et al., Bioorg. Med. Chem.Lett., 2002, 12, 663-666), a series of 4-aryl-pyridines containing botha 3-[(1R)-hydroxyethyl] and a 2′-hydroxy group (Ladouceur et al.,Bioorg. Med. Chem. Lett., 2002, 12, 3421-3424), a series of5-hydroxyalkyl-4-phenylpyridines (Ladouceur et al., Bioorg. Med. Chem.Lett., 2002, 12, 461-464), a series of triarylimidazoles (Chang et al.,Bioorg. Med. Chem. Lett., 2001, 11, 2549-2553), a series of2-pyridyl-3,5-diaryl pyrroles (de Laszlo et al., Bioorg. Med. Chem.Lett., 1999, 9, 641-646), several substituted benzimidazoles (Madsen etal., J. Med. Chem., 1998, 41, 5150-5157), and a series ofpyrrolo[1,2-a]quinoxalines (Guillon et al., Eur. J. Med. Chem., 1998,33, 293-308).

There remains a long felt need for additional agents capable ofeffectively inhibiting glucagon receptor function. Antisense technologyis an effective means for reducing the expression of specific geneproducts and has proven to be uniquely useful in a number oftherapeutic, diagnostic, and research applications. The presentinvention provides compositions and methods for modulating glucagonreceptor expression.

SUMMARY OF THE INVENTION

The present invention is directed to compounds, especially nucleic acidand nucleic acid-like oligomers, which are targeted to a nucleic acidencoding glucagon receptor, and which modulate the expression ofglucagon receptor. Pharmaceutical and other compositions comprising thecompounds of the invention are also provided. Further provided aremethods of screening for modulators of glucagon receptor and methods ofmodulating the expression of glucagon receptor in cells, tissues oranimals comprising contacting said cells, tissues or animals with one ormore of the compounds or compositions of the invention. Methods oftreating an animal, particularly a human, suspected of having or beingprone to a disease or condition associated with expression of glucagonreceptor are also set forth herein. Such methods comprise administeringa therapeutically or prophylactically effective amount of one or more ofthe compounds or compositions of the invention to the person in need oftreatment.

DETAILED DESCRIPTION OF THE INVENTION A. Overview of the Invention

The present invention employs compounds, preferably oligonucleotides andsimilar species for use in modulating the function or effect of nucleicacid molecules encoding glucagon receptor. This is accomplished byproviding oligonucleotides which specifically hybridize with one or morenucleic acid molecules encoding glucagon receptor. As used herein, theterms “target nucleic acid” and “nucleic acid molecule encoding glucagonreceptor” have been used for convenience to encompass DNA encodingglucagon receptor, RNA (including pre-mRNA and mRNA or portions thereof)transcribed from such DNA, and also cDNA derived from such RNA. Thehybridization of a compound of this invention with its target nucleicacid is generally referred to as “antisense”. Consequently, thepreferred mechanism believed to be included in the practice of somepreferred embodiments of the invention is referred to herein as“antisense inhibition.” Such antisense inhibition is typically basedupon hydrogen bonding-based hybridization of oligonucleotide strands orsegments such that at least one strand or segment is cleaved, degraded,or otherwise rendered inoperable. In this regard, it is presentlypreferred to target specific nucleic acid molecules and their functionsfor such antisense inhibition.

The functions of DNA to be interfered with can include replication andtranscription. Replication and transcription, for example, can be froman endogenous cellular template, a vector, a plasmid construct orotherwise. The functions of RNA to be interfered with can includefunctions such as translocation of the RNA to a site of proteintranslation, translocation of the RNA to sites within the cell which aredistant from the site of RNA synthesis, translation of protein from theRNA, splicing of the RNA to yield one or more RNA species, and catalyticactivity or complex formation involving the RNA which may be engaged inor facilitated by the RNA. One preferred result of such interferencewith target nucleic acid function is modulation of the expression ofglucagon receptor. In the context of the present invention, “modulation”and “modulation of expression” mean either an increase (stimulation) ora decrease (inhibition) in the amount or levels of a nucleic acidmolecule encoding the gene, e.g., DNA or RNA. Inhibition is often thepreferred form of modulation of expression and mRNA is often a preferredtarget nucleic acid.

In the context of this invention, “hybridization” means the pairing ofcomplementary strands of oligomeric compounds. In the present invention,the preferred mechanism of pairing involves hydrogen bonding, which maybe Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding,between complementary nucleoside or nucleotide bases (nucleobases) ofthe strands of oligomeric compounds. For example, adenine and thymineare complementary nucleobases which pair through the formation ofhydrogen bonds. Hybridization can occur under varying circumstances.

An antisense compound is specifically hybridizable when binding of thecompound to the target nucleic acid interferes with the normal functionof the target nucleic acid to cause a loss of activity, and there is asufficient degree of complementarity to avoid non-specific binding ofthe antisense compound to non-target nucleic acid sequences underconditions in which specific binding is desired, i.e., underphysiological conditions in the case of in vivo assays or therapeutictreatment, and under conditions in which assays are performed in thecase of in vitro assays.

In the present invention the phrase “stringent hybridization conditions”or “stringent conditions” refers to conditions under which a compound ofthe invention will hybridize to its target sequence, but to a minimalnumber of other sequences. Stringent conditions are sequence-dependentand will be different in different circumstances and in the context ofthis invention, “stringent conditions” under which oligomeric compoundshybridize to a target sequence are determined by the nature andcomposition of the oligomeric compounds and the assays in which they arebeing investigated.

“Complementary,” as used herein, refers to the capacity for precisepairing between two nucleobases of an oligomeric compound. For example,if a nucleobase at a certain position of an oligonucleotide (anoligomeric compound), is capable of hydrogen bonding with a nucleobaseat a certain position of a target nucleic acid, said target nucleic acidbeing a DNA, RNA, or oligonucleotide molecule, then the position ofhydrogen bonding between the oligonucleotide and the target nucleic acidis considered to be a complementary position. The oligonucleotide andthe further DNA, RNA, or oligonucleotide molecule are complementary toeach other when a sufficient number of complementary positions in eachmolecule are occupied by nucleobases which can hydrogen bond with eachother. Thus, “specifically hybridizable” and “complementary” are termswhich are used to indicate a sufficient degree of precise pairing orcomplementarity over a sufficient number of nucleobases such that stableand specific binding occurs between the oligonucleotide and a targetnucleic acid.

It is understood in the art that the sequence of an antisense compoundneed not be 100% complementary to that of its target nucleic acid to bespecifically hybridizable. Moreover, an oligonucleotide may hybridizeover one or more segments such that intervening or adjacent segments arenot involved in the hybridization event (e.g., a loop structure orhairpin structure). It is preferred that the antisense compounds of thepresent invention comprise at least 70% sequence complementarity to atarget region within the target nucleic acid, more preferably that theycomprise 90% sequence complementarity and even more preferably comprise95% sequence complementarity to the target region within the targetnucleic acid sequence to which they are targeted. For example, anantisense compound in which 18 of 20 nucleobases of the antisensecompound are complementary to a target region, and would thereforespecifically hybridize, would represent 90 percent complementarity. Inthis example, the remaining noncomplementary nucleobases may beclustered or interspersed with complementary nucleobases and need not becontiguous to each other or to complementary nucleobases. As such, anantisense compound which is 18 nucleobases in length having 4 (four)noncomplementary nucleobases which are flanked by two regions ofcomplete complementarity with the target nucleic acid would have 77.8%overall complementarity with the target nucleic acid and would thus fallwithin the scope of the present invention. Percent complementarity of anantisense compound with a region of a target nucleic acid can bedetermined routinely using BLAST programs (basic local alignment searchtools) and PowerBLAST programs known in the art (Altschul et al., J.Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7,649-656).

B. Compounds of the Invention

According to the present invention, compounds include antisenseoligomeric compounds, antisense oligonucleotides, ribozymes, externalguide sequence (EGS) oligonucleotides, alternate splicers, primers,probes, and other oligomeric compounds which hybridize to at least aportion of the target nucleic acid. As such, these compounds may beintroduced in the form of single-stranded, double-stranded, circular orhairpin oligomeric compounds and may contain structural elements such asinternal or terminal bulges or loops. Once introduced to a system, thecompounds of the invention may elicit the action of one or more enzymesor structural proteins to effect modification of the target nucleicacid.

One non-limiting example of such an enzyme is RNAse H, a cellularendonuclease which cleaves the RNA strand of an RNA:DNA duplex. It isknown in the art that single-stranded antisense compounds which are“DNA-like” elicit RNAse H. Activation of RNase H, therefore, results incleavage of the RNA target, thereby greatly enhancing the efficiency ofoligonucleotide-mediated inhibition of gene expression. Similar roleshave been postulated for other ribonucleases such as those in the RNaseIII and ribonuclease L family of enzymes.

While the preferred form of antisense compound is a single-strandedantisense oligonucleotide, in many species the introduction ofdouble-stranded structures, such as double-stranded RNA (dsRNA)molecules, has been shown to induce potent and specificantisense-mediated reduction of the function of a gene or its associatedgene products. This phenomenon occurs in both plants and animals and isbelieved to have an evolutionary connection to viral defense andtransposon silencing.

The first evidence that dsRNA could lead to gene silencing in animalscame in 1995 from work in the nematode, Caenorhabditis elegans (Guo andKempheus, Cell, 1995, 81, 611-620).

Montgomery et al. have shown that the primary interference effects ofdsRNA are posttranscriptional (Montgomery et al., Proc. Natl. Acad. Sci.USA, 1998, 95, 15502-15507). The posttranscriptional antisense mechanismdefined in Caenorhabditis elegans resulting from exposure todouble-stranded RNA (dsRNA) has since been designated RNA interference(RNAi). This term has been generalized to mean antisense-mediated genesilencing involving the introduction of dsRNA leading to thesequence-specific reduction of endogenous targeted mRNA levels (Fire etal., Nature, 1998, 391, 806-811). Recently, it has been shown that itis, in fact, the single-stranded RNA oligomers of antisense polarity ofthe dsRNAs which are the potent inducers of RNAi (Tijsterman et al.,Science, 2002, 295, 694-697).

In the context of this invention, the term “oligomeric compound” refersto a polymer or oligomer comprising a plurality of monomeric units. Inthe context of this invention, the term “oligonucleotide” refers to anoligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid(DNA) or mimetics, chimeras, analogs and homologs thereof. This termincludes oligonucleotides composed of naturally occurring nucleobases,sugars and covalent internucleoside (backbone) linkages as well asoligonucleotides having non-naturally occurring portions which functionsimilarly. Such modified or substituted oligonucleotides are oftenpreferred over native forms because of desirable properties such as, forexample, enhanced cellular uptake, enhanced affinity for a targetnucleic acid and increased stability in the presence of nucleases.

While oligonucleotides are a preferred form of the compounds of thisinvention, the present invention comprehends other families of compoundsas well, including but not limited to oligonucleotide analogs andmimetics such as those described herein.

The compounds in accordance with this invention preferably comprise fromabout 8 to about 80 nucleobases (i.e. from about 8 to about 80 linkednucleosides). One of ordinary skill in the art will appreciate that theinvention embodies compounds of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleobases in length.

In one preferred embodiment, the compounds of the invention are 12 to 50nucleobases in length. One having ordinary skill in the art willappreciate that this embodies compounds of 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleobases inlength.

In another preferred embodiment, the compounds of the invention are 15to 30 nucleobases in length. One having ordinary skill in the art willappreciate that this embodies compounds of 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleobases in length.

Particularly preferred compounds are oligonucleotides from about 12 toabout 50 nucleobases, even more preferably those comprising from about15 to about 30 nucleobases.

Antisense compounds 8-80 nucleobases in length comprising a stretch ofat least eight (8) consecutive nucleobases selected from within theillustrative antisense compounds are considered to be suitable antisensecompounds as well.

Exemplary preferred antisense compounds include oligonucleotidesequences that comprise at least the 8 consecutive nucleobases from the5′-terminus of one of the illustrative preferred antisense compounds(the remaining nucleobases being a consecutive stretch of the sameoligonucleotide beginning immediately upstream of the 5′-terminus of theantisense compound which is specifically hybridizable to the targetnucleic acid and continuing until the oligonucleotide contains about 8to about 80 nucleobases). Similarly preferred antisense compounds arerepresented by oligonucleotide sequences that comprise at least the 8consecutive nucleobases from the 3′-terminus of one of the illustrativepreferred antisense compounds (the remaining nucleobases being aconsecutive stretch of the same oligonucleotide beginning immediatelydownstream of the 3′-terminus of the antisense compound which isspecifically hybridizable to the target nucleic acid and continuinguntil the oligonucleotide contains about 8 to about 80 nucleobases). Onehaving skill in the art armed with the preferred antisense compoundsillustrated herein will be able, without undue experimentation, toidentify further preferred antisense compounds.

C. Targets of the Invention

“Targeting” an antisense compound to a particular nucleic acid molecule,in the context of this invention, can be a multistep process. Theprocess usually begins with the identification of a target nucleic acidwhose function is to be modulated. This target nucleic acid may be, forexample, a cellular gene (or mRNA transcribed from the gene) whoseexpression is associated with a particular disorder or disease state, ora nucleic acid molecule from an infectious agent. In the presentinvention, the target nucleic acid encodes glucagon receptor.

The targeting process usually also includes determination of at leastone target region, segment, or site within the target nucleic acid forthe antisense interaction to occur such that the desired effect, e.g.,modulation of expression, will result. Within the context of the presentinvention, the term “region” is defined as a portion of the targetnucleic acid having at least one identifiable structure, function, orcharacteristic. Within regions of target nucleic acids are segments.“Segments” are defined as smaller or sub-portions of regions within atarget nucleic acid. “Sites,” as used in the present invention, aredefined as positions within a target nucleic acid.

Since, as is known in the art, the translation initiation codon istypically 5′-AUG (in transcribed mRNA molecules; 5′-ATG in thecorresponding DNA molecule), the translation initiation codon is alsoreferred to as the “AUG codon,” the “start codon” or the “AUG startcodon”. A minority of genes have a translation initiation codon havingthe RNA sequence 5′-GUG, 5′-UUG or 5′-CUG, and 5′-AUA, 5′-ACG and 5′-CUGhave been shown to function in vivo. Thus, the terms “translationinitiation codon” and “start codon” can encompass many codon sequences,even though the initiator amino acid in each instance is typicallymethionine (in eukaryotes) or formylmethionine (in prokaryotes). It isalso known in the art that eukaryotic and prokaryotic genes may have twoor more alternative start codons, any one of which may be preferentiallyutilized for translation initiation in a particular cell type or tissue,or under a particular set of conditions. In the context of theinvention, “start codon” and “translation initiation codon” refer to thecodon or codons that are used in vivo to initiate translation of an mRNAtranscribed from a gene encoding glucagon receptor, regardless of thesequence(s) of such codons. It is also known in the art that atranslation termination codon (or “stop codon”) of a gene may have oneof three sequences, i.e., 5′-UAA, 5′-UAG and 5′-UGA (the correspondingDNA sequences are 5′-TAA, 5′-TAG and 5′-TGA, respectively).

The terms “start codon region” and “translation initiation codon region”refer to a portion of such an mRNA or gene that encompasses from about25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or3′) from a translation initiation codon. Similarly, the terms “stopcodon region” and “translation termination codon region” refer to aportion of such an mRNA or gene that encompasses from about 25 to about50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from atranslation termination codon. Consequently, the “start codon region”(or “translation initiation codon region”) and the “stop codon region”(or “translation termination codon region”) are all regions which may betargeted effectively with the antisense compounds of the presentinvention.

The open reading frame (ORF) or “coding region,” which is known in theart to refer to the region between the translation initiation codon andthe translation termination codon, is also a region which may betargeted effectively. Within the context of the present invention, apreferred region is the intragenic region encompassing the translationinitiation or termination codon of the open reading frame (ORF) of agene.

Other target regions include the 5′ untranslated region (5′UTR), knownin the art to refer to the portion of an mRNA in the 5′ direction fromthe translation initiation codon, and thus including nucleotides betweenthe 5′ cap site and the translation initiation codon of an mRNA (orcorresponding nucleotides on the gene), and the 3′ untranslated region(3′UTR), known in the art to refer to the portion of an mRNA in the 3′direction from the translation termination codon, and thus includingnucleotides between the translation termination codon and 3′ end of anmRNA (or corresponding nucleotides on the gene). The 5′ cap site of anmRNA comprises an N7-methylated guanosine residue joined to the 5′-mostresidue of the mRNA via a 5′-5′ triphosphate linkage. The 5′ cap regionof an mRNA is considered to include the 5′ cap structure itself as wellas the first 50 nucleotides adjacent to the cap site. It is alsopreferred to target the 5′ cap region.

Although some eukaryotic mRNA transcripts are directly translated, manycontain one or more regions, known as “introns,” which are excised froma transcript before it is translated. The remaining (and thereforetranslated) regions are known as “exons” and are spliced together toform a continuous mRNA sequence. Targeting splice sites, i.e.,intron-exon junctions or exon-intron junctions, may also be particularlyuseful in situations where aberrant splicing is implicated in disease,or where an overproduction of a particular splice product is implicatedin disease. Aberrant fusion junctions due to rearrangements or deletionsare also preferred target sites. mRNA transcripts produced via theprocess of splicing of two (or more) mRNAs from different gene sourcesare known as “fusion transcripts”. It is also known that introns can beeffectively targeted using antisense compounds targeted to, for example,DNA or pre-mRNA.

It is also known in the art that alternative RNA transcripts can beproduced from the same genomic region of DNA. These alternativetranscripts are generally known as “variants”. More specifically,“pre-mRNA variants” are transcripts produced from the same genomic DNAthat differ from other transcripts produced from the same genomic DNA ineither their start or stop position and contain both intronic and exonicsequence.

Upon excision of one or more exon or intron regions, or portions thereofduring splicing, pre-mRNA variants produce smaller “mRNA variants”.Consequently, mRNA variants are processed pre-mRNA variants and eachunique pre-mRNA variant must always produce a unique mRNA variant as aresult of splicing. These mRNA variants are also known as “alternativesplice variants”. If no splicing of the pre-mRNA variant occurs then thepre-mRNA variant is identical to the mRNA variant.

It is also known in the art that variants can be produced through theuse of alternative signals to start or stop transcription and thatpre-mRNAs and mRNAs can possess more that one start codon or stop codon.Variants that originate from a pre-mRNA or mRNA that use alternativestart codons are known as “alternative start variants” of that pre-mRNAor mRNA. Those transcripts that use an alternative stop codon are knownas “alternative stop variants” of that pre-mRNA or mRNA. One specifictype of alternative stop variant is the “polyA variant” in which themultiple transcripts produced result from the alternative selection ofone of the “polyA stop signals” by the transcription machinery, therebyproducing transcripts that terminate at unique polyA sites. Within thecontext of the invention, the types of variants described herein arealso preferred target nucleic acids.

The locations on the target nucleic acid to which the preferredantisense compounds hybridize are hereinbelow referred to as “preferredtarget segments.” As used herein the term “preferred target segment” isdefined as at least an 8-nucleobase portion of a target region to whichan active antisense compound is targeted. While not wishing to be boundby theory, it is presently believed that these target segments representportions of the target nucleic acid which are accessible forhybridization.

While the specific sequences of certain preferred target segments areset forth herein, one of skill in the art will recognize that theseserve to illustrate and describe particular embodiments within the scopeof the present invention. Additional preferred target segments may beidentified by one having ordinary skill.

Target segments 8-80 nucleobases in length comprising a stretch of atleast eight (8) consecutive nucleobases selected from within theillustrative preferred target segments are considered to be suitable fortargeting as well.

Target segments can include DNA or RNA sequences that comprise at leastthe 8 consecutive nucleobases from the 5′-terminus of one of theillustrative preferred target segments (the remaining nucleobases beinga consecutive stretch of the same DNA or RNA beginning immediatelyupstream of the 5′-terminus of the target segment and continuing untilthe DNA or RNA contains about 8 to about 80 nucleobases). Similarlypreferred target segments are represented by DNA or RNA sequences thatcomprise at least the 8 consecutive nucleobases from the 3′-terminus ofone of the illustrative preferred target segments (the remainingnucleobases being a consecutive stretch of the same DNA or RNA beginningimmediately downstream of the 3′-terminus of the target segment andcontinuing until the DNA or RNA contains about 8 to about 80nucleobases). One having skill in the art armed with the preferredtarget segments illustrated herein will be able, without undueexperimentation, to identify further preferred target segments.

Once one or more target regions, segments or sites have been identified,antisense compounds are chosen which are sufficiently complementary tothe target, i.e., hybridize sufficiently well and with sufficientspecificity, to give the desired effect.

D. Screening and Target Validation

In a further embodiment, the “preferred target segments” identifiedherein may be employed in a screen for additional compounds thatmodulate the expression of glucagon receptor. “Modulators” are thosecompounds that decrease or increase the expression of a nucleic acidmolecule encoding glucagon receptor and which comprise at least an8-nucleobase portion which is complementary to a preferred targetsegment. The screening method comprises the steps of contacting apreferred target segment of a nucleic acid molecule encoding glucagonreceptor with one or more candidate modulators, and selecting for one ormore candidate modulators which decrease or increase the expression of anucleic acid molecule encoding glucagon receptor. Once it is shown thatthe candidate modulator or modulators are capable of modulating (e.g.either decreasing or increasing) the expression of a nucleic acidmolecule encoding glucagon receptor, the modulator may then be employedin further investigative studies of the function of glucagon receptor,or for use as a research, diagnostic, or therapeutic agent in accordancewith the present invention.

The preferred target segments of the present invention may be also becombined with their respective complementary antisense compounds of thepresent invention to form stabilized double-stranded (duplexed)oligonucleotides.

Such double stranded oligonucleotide moieties have been shown in the artto modulate target expression and regulate translation as well as RNAprocessing via an antisense mechanism. Moreover, the double-strandedmoieties may be subject to chemical modifications (Fire et al., Nature,1998, 391, 806-811; Timmons and Fire, Nature 1998, 395, 854; Timmons etal., Gene, 2001, 263, 103-112; Tabara et al., Science, 1998, 282,430-431; Montgomery et al., Proc. Natl. Acad. Sci. USA, 1998, 95,15502-15507; Tuschl et al., Genes Dev., 1999, 13, 3191-3197; Elbashir etal., Nature, 2001, 411, 494-498; Elbashir et al., Genes Dev. 2001, 15,188-200). For example, such double-stranded moieties have been shown toinhibit the target by the classical hybridization of antisense strand ofthe duplex to the target, thereby triggering enzymatic degradation ofthe target (Tijsterman et al., Science, 2002, 295, 694-697).

The compounds of the present invention can also be applied in the areasof drug discovery and target validation. The present inventioncomprehends the use of the compounds and preferred target segmentsidentified herein in drug discovery efforts to elucidate relationshipsthat exist between glucagon receptor and a disease state, phenotype, orcondition. These methods include detecting or modulating glucagonreceptor comprising contacting a sample, tissue, cell, or organism withthe compounds of the present invention, measuring the nucleic acid orprotein level of glucagon receptor and/or a related phenotypic orchemical endpoint at some time after treatment, and optionally comparingthe measured value to a non-treated sample or sample treated with afurther compound of the invention. These methods can also be performedin parallel or in combination with other experiments to determine thefunction of unknown genes for the process of target validation or todetermine the validity of a particular gene product as a target fortreatment or prevention of a particular disease, condition, orphenotype.

E. Kits, Research Reagents, Diagnostics, and Therapeutics

The compounds of the present invention can be utilized for diagnostics,therapeutics (including prophylaxis) and as research reagents and kits.Furthermore, antisense oligonucleotides, which are able to inhibit geneexpression with exquisite specificity, are often used by those ofordinary skill to elucidate the function of particular genes or todistinguish between functions of various members of a biologicalpathway.

For use in kits and diagnostics, the compounds of the present invention,either alone or in combination with other compounds or therapeutics, canbe used as tools in differential and/or combinatorial analyses toelucidate expression patterns of a portion or the entire complement ofgenes expressed within cells and tissues.

As one nonlimiting example, expression patterns within cells or tissuestreated with one or more antisense compounds are compared to controlcells or tissues not treated with antisense compounds and the patternsproduced are analyzed for differential levels of gene expression as theypertain, for example, to disease association, signaling pathway,cellular localization, expression level, size, structure or function ofthe genes examined. These analyses can be performed on stimulated orunstimulated cells and in the presence or absence of other compoundswhich affect expression patterns.

Examples of methods of gene expression analysis known in the art includeDNA arrays or microarrays (Brazma and Vilo, FEBS Lett., 2000, 480,17-24; Celis, et al., FEBS Lett., 2000, 480, 2-16), SAGE (serialanalysis of gene expression) (Madden, et al., Drug Discov. Today, 2000,5, 415-425), READS (restriction enzyme amplification of digested cDNAs)(Prashar and Weissman, Methods Enzymol., 1999, 303, 258-72), TOGA (totalgene expression analysis) (Sutcliffe, et al., Proc. Natl. Acad. Sci.U.S.A., 2000, 97, 1976-81), protein arrays and proteomics (Celis, etal., FEBS Lett., 2000, 480, 2-16; Jungblut, et al., Electrophoresis,1999, 20, 2100-10), expressed sequence tag (EST) sequencing (Celis, etal., FEBS Lett., 2000, 480, 2-16; Larsson, et al., J. Biotechnol., 2000,80, 143-57), subtractive RNA fingerprinting (SuRF) (Fuchs, et al., Anal.Biochem., 2000, 286, 91-98; Larson, et al., Cytometry, 2000, 41,203-208), subtractive cloning, differential display (DD) (Jurecic andBelmont, Curr. Opin. Microbiol., 2000, 3, 316-21), comparative genomichybridization (Carulli, et al., J. Cell Biochem. Suppl., 1998, 31,286-96), FISH (fluorescent in situ hybridization) techniques (Going andGusterson, Eur. J. Cancer, 1999, 35, 1895-904) and mass spectrometrymethods (To, Comb. Chem. High Throughput Screen, 2000, 3, 235-41).

The compounds of the invention are useful for research and diagnostics,because these compounds hybridize to nucleic acids encoding glucagonreceptor. For example, oligonucleotides that are shown to hybridize withsuch efficiency and under such conditions as disclosed herein as to beeffective glucagon receptor inhibitors will also be effective primers orprobes under conditions favoring gene amplification or detection,respectively. These primers and probes are useful in methods requiringthe specific detection of nucleic acid molecules encoding glucagonreceptor and in the amplification of said nucleic acid molecules fordetection or for use in further studies of glucagon receptor.Hybridization of the antisense oligonucleotides, particularly theprimers and probes, of the invention with a nucleic acid encodingglucagon receptor can be detected by means known in the art. Such meansmay include conjugation of an enzyme to the oligonucleotide,radiolabelling of the oligonucleotide or any other suitable detectionmeans. Kits using such detection means for detecting the level ofglucagon receptor in a sample may also be prepared.

The specificity and sensitivity of antisense is also harnessed by thoseof skill in the art for therapeutic uses. Antisense compounds have beenemployed as therapeutic moieties in the treatment of disease states inanimals, including humans. Antisense oligonucleotide drugs, includingribozymes, have been safely and effectively administered to humans andnumerous clinical trials are presently underway. It is thus establishedthat antisense compounds can be useful therapeutic modalities that canbe configured to be useful in treatment regimes for the treatment ofcells, tissues and animals, especially humans.

For therapeutics, an animal, preferably a human, suspected of having adisease or disorder which can be treated by modulating the expression ofglucagon receptor is treated by administering antisense compounds inaccordance with this invention. For example, in one non-limitingembodiment, the methods comprise the step of administering to an animala therapeutically effective amount of a glucagon receptor inhibitor. Theglucagon receptor inhibitors of the present invention effectivelyinhibit the activity of the glucagon receptor protein or inhibit theexpression of the glucagon receptor protein. In one embodiment, theactivity or expression of glucagon receptor in an animal is inhibited byabout 10%. Preferably, the activity or expression of glucagon receptorin an animal is inhibited by about 30%. More preferably, the activity orexpression of glucagon receptor in an animal is inhibited by 50% ormore. Because the compounds herein are inhibitors of glucagon receptor,they are believed to be useful in lowering blood glucose, for example,and in treating conditions associated with glucagon receptor activity,such as high blood glucose and other metabolic conditions such asdiabetes (including Type 2 diabetes), obesity, and insulin resistance.

The reduction of the expression of glucagon receptor may be measured,for example, in blood, plasma, serum, adipose tissue, liver or any otherbody fluid, tissue or organ of the animal. Preferably, the cellscontained within said fluids, tissues or organs being analyzed contain anucleic acid molecule encoding glucagon receptor protein and/or theglucagon receptor protein itself.

The compounds of the invention can be utilized in pharmaceuticalcompositions by adding an effective amount of a compound to a suitablepharmaceutically acceptable diluent or carrier. Use of the compounds andmethods of the invention may also be useful prophylactically.

F. Modifications

As is known in the art, a nucleoside is a base-sugar combination. Thebase portion of the nucleoside is normally a heterocyclic base. The twomost common classes of such heterocyclic bases are the purines and thepyrimidines. Nucleotides are nucleosides that further include aphosphate group covalently linked to the sugar portion of thenucleoside. For those nucleosides that include a pentofuranosyl sugar,the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxylmoiety of the sugar. In forming oligonucleotides, the phosphate groupscovalently link adjacent nucleosides to one another to form a linearpolymeric compound. In turn, the respective ends of this linearpolymeric compound can be further joined to form a circular compound,however, linear compounds are generally preferred. In addition, linearcompounds may have internal nucleobase complementarity and may thereforefold in a manner as to produce a fully or partially double-strandedcompound. Within oligonucleotides, the phosphate groups are commonlyreferred to as forming the internucleoside backbone of theoligonucleotide. The normal linkage or backbone of RNA and DNA is a 3′to 5′ phosphodiester linkage.

Modified Internucleoside Linkages (Backbones)

Specific examples of preferred antisense compounds useful in thisinvention include oligonucleotides containing modified backbones ornon-natural internucleoside linkages. As defined in this specification,oligonucleotides having modified backbones include those that retain aphosphorus atom in the backbone and those that do not have a phosphorusatom in the backbone. For the purposes of this specification, and assometimes referenced in the art, modified oligonucleotides that do nothave a phosphorus atom in their internucleoside backbone can also beconsidered to be oligonucleosides.

Preferred modified oligonucleotide backbones containing a phosphorusatom therein include, for example, phosphorothioates, chiralphosphorothioates, phosphorodithioates, phosphotriesters,aminoalkylphosphotriesters, methyl and other alkyl phosphonatesincluding 3′-alkylene phosphonates, 5′-alkylene phosphonates and chiralphosphonates, phosphinates, phosphoramidates including 3′-aminophosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphatesand boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogsof these, and those having inverted polarity wherein one or moreinternucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage.Preferred oligonucleotides having inverted polarity comprise a single 3′to 3′ linkage at the 3′-most internucleotide linkage i.e. a singleinverted nucleoside residue which may be abasic (the nucleobase ismissing or has a hydroxyl group in place thereof). Various salts, mixedsalts and free acid forms are also included.

Representative United States patents that teach the preparation of theabove phosphorus-containing linkages include, but are not limited to,U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799;5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697 and5,625,050, certain of which are commonly owned with this application,and each of which is herein incorporated by reference.

Preferred modified oligonucleotide backbones that do not include aphosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; riboacetyl backbones; alkene containingbackbones; sulfamate backbones; methyleneimino and methylenehydrazinobackbones; sulfonate and sulfonamide backbones; amide backbones; andothers having mixed N, O, S and CH₂ component parts.

Representative United States patents that teach the preparation of theabove oligonucleotides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, certain ofwhich are commonly owned with this application, and each of which isherein incorporated by reference.

Modified Sugar and Internucleoside Linkages-Mimetics

In other preferred oligonucleotide mimetics, both the sugar and theinternucleoside linkage (i.e. the backbone), of the nucleotide units arereplaced with novel groups. The nucleobase units are maintained forhybridization with an appropriate target nucleic acid. One suchcompound, an oligonucleotide mimetic that has been shown to haveexcellent hybridization properties, is referred to as a peptide nucleicacid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotideis replaced with an amide containing backbone, in particular anaminoethylglycine backbone. The nucleobases are retained and are bounddirectly or indirectly to aza nitrogen atoms of the amide portion of thebackbone. Representative United States patents that teach thepreparation of PNA compounds include, but are not limited to, U.S. Pat.Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is hereinincorporated by reference. Further teaching of PNA compounds can befound in Nielsen et al., Science, 1991, 254, 1497-1500.

Preferred embodiments of the invention are oligonucleotides withphosphorothioate backbones. Also preferred are oligonucleosides withheteroatom backbones, and in particular —CH₂—NH—O—CH₂—,—CH₂—N(CH₃)—O—CH₂— [known as a methylene (methylimino) or MMI backbone],—CH₂—O—N(CH₃)—CH₂—, —CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂—[wherein the native phosphodiester backbone is represented as—O—P—O—CH₂—] of the above referenced U.S. Pat. No. 5,489,677, and theamide backbones of the above referenced U.S. Pat. No. 5,602,240. Alsopreferred are oligonucleotides having morpholino backbone structures ofthe above-referenced U.S. Pat. No. 5,034,506.

Modified Sugars

Modified oligonucleotides may also contain one or more substituted sugarmoieties. Preferred oligonucleotides comprise one of the following atthe 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynylmay be substituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyland alkynyl. Particularly preferred are O[(CH₂)_(n)O]_(m)CH₃,O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, andO(CH₂)_(n)ON[(CH₂)_(n)CH₃]₂, where n and m are from 1 to about 10. Otherpreferred oligonucleotides comprise one of the following at the 2′position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkenyl,alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl,Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl,heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl,an RNA cleaving group, a reporter group, an intercalator, a group forimproving the pharmacokinetic properties of an oligonucleotide, or agroup for improving the pharmacodynamic properties of anoligonucleotide, and other substituents having similar properties. Apreferred modification includes 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, alsoknown as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim.Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A furtherpreferred modification includes 2′-dimethylaminooxyethoxy, i.e., aO(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described in exampleshereinbelow, and 2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethyl-amino-ethoxy-ethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₃)₂, also described in examples hereinbelow.

Other preferred modifications include 2′-methoxy (2′-O—CH₃),2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂), 2′-allyl (2′-CH₂—CH═CH₂), 2′-O-allyl(2′-O—CH₂—CH═CH₂) and 2′-fluoro (2′-F). The 2′-modification may be inthe arabino (up) position or ribo (down) position. A preferred2′-arabino modification is 2′-F. Similar modifications may also be madeat other positions on the oligonucleotide, particularly the 3′ positionof the sugar on the 3′ terminal nucleotide or in 2′-5′ linkedoligonucleotides and the 5′ position of 5′ terminal nucleotide.Oligonucleotides may also have sugar mimetics such as cyclobutylmoieties in place of the pentofuranosyl sugar. Representative UnitedStates patents that teach the preparation of such modified sugarstructures include, but are not limited to, U.S. Pat. Nos. 4,981,957;5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786;5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909;5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633;5,792,747; and 5,700,920, certain of which are commonly owned with theinstant application, and each of which is herein incorporated byreference in its entirety.

A further preferred modification of the sugar includes Locked NucleicAcids (LNAs) in which the 2′-hydroxyl group is linked to the 3′ or 4′carbon atom of the sugar ring, thereby forming a bicyclic sugar moiety.The linkage is preferably a methylene (—CH₂—)_(n) group bridging the 2′oxygen atom and the 4′ carbon atom wherein n is 1 or 2. LNAs andpreparation thereof are described in WO 98/39352 and WO 99/14226.

Natural and Modified Nucleobases

Oligonucleotides may also include nucleobase (often referred to in theart simply as “base”) modifications or substitutions. As used herein,“unmodified” or “natural” nucleobases include the purine bases adenine(A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C)and uracil (U). Modified nucleobases include other synthetic and naturalnucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine,xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,5-halouracil and cytosine, 5-propynyl (—C≡C—CH₃) uracil and cytosine andother alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosineand thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino,8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines andguanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and3-deazaadenine. Further modified nucleobases include tricyclicpyrimidines such as phenoxazinecytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazinecytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps suchas a substituted phenoxazine cytidine (e.g.9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazolecytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine(H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobasesmay also include those in which the purine or pyrimidine base isreplaced with other heterocycles, for example 7-deaza-adenine,7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobasesinclude those disclosed in U.S. Pat. No. 3,687,808, those disclosed inThe Concise Encyclopedia Of Polymer Science And Engineering, pages858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosedby Englisch et al., Angewandte Chemie, International Edition, 1991, 30,613, and those disclosed by Sanghvi, Y. S., Chapter 15, AntisenseResearch and Applications, pages 289-302, Crooke, S. T. and Lebleu, B.,ed., CRC Press, 1993. Certain of these nucleobases are particularlyuseful for increasing the binding affinity of the compounds of theinvention. These include 5-substituted pyrimidines, 6-azapyrimidines andN-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine,5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutionshave been shown to increase nucleic acid duplex stability by 0.6-1.2° C.and are presently preferred base substitutions.

Representative United States patents that teach the preparation ofcertain of the above noted modified nucleobases as well as othermodified nucleobases include, but are not limited to, the above notedU.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302;5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255;5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121,5,596,091; 5,614,617; 5,645,985; 5,830,653; 5,763,588; 6,005,096; and5,681,941, certain of which are commonly owned with the instantapplication, and each of which is herein incorporated by reference, andU.S. Pat. No. 5,750,692, which is commonly owned with the instantapplication and also herein incorporated by reference.

Conjugates

Another modification of the oligonucleotides of the invention involveschemically linking to the oligonucleotide one or more moieties orconjugates which enhance the activity, cellular distribution or cellularuptake of the oligonucleotide. These moieties or conjugates can includeconjugate groups covalently bound to functional groups such as primaryor secondary hydroxyl groups. Conjugate groups of the invention includeintercalators, reporter molecules, polyamines, polyamides, polyethyleneglycols, polyethers, groups that enhance the pharmacodynamic propertiesof oligomers, and groups that enhance the pharmacokinetic properties ofoligomers. Typical conjugate groups include cholesterols, lipids,phospholipids, biotin, phenazine, folate, phenan-thridine,anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes.Groups that enhance the pharmacodynamic properties, in the context ofthis invention, include groups that improve uptake, enhance resistanceto degradation, and/or strengthen sequence-specific hybridization withthe target nucleic acid. Groups that enhance the pharmacokineticproperties, in the context of this invention, include groups thatimprove uptake, distribution, metabolism or excretion of the compoundsof the present invention. Representative conjugate groups are disclosedin International Patent Application PCT/US92/09196, filed Oct. 23, 1992,and U.S. Pat. No. 6,287,860, the entire disclosure of which areincorporated herein by reference. Conjugate moieties include but are notlimited to lipid moieties such as a cholesterol moiety, cholic acid, athioether, e.g., hexyl-5-tritylthiol, a thiocholesterol, an aliphaticchain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or apolyethylene glycol chain, or adamantane acetic acid, a palmityl moiety,or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety.Oligonucleotides of the invention may also be conjugated to active drugsubstances, for example, aspirin, warfarin, phenylbutazone, ibuprofen,suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen,dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinicacid, a benzothiadiazide, chlorothiazide, a diazepine, indomethicin, abarbiturate, a cephalosporin, a sulfa drug, an antidiabetic, anantibacterial or an antibiotic. Oligonucleotide-drug conjugates andtheir preparation are described in U.S. patent application Ser. No.09/334,130 (filed Jun. 15, 1999) which is incorporated herein byreference in its entirety.

Representative United States patents that teach the preparation of sucholigonucleotide conjugates include, but are not limited to, U.S. Pat.Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, certain ofwhich are commonly owned with the instant application, and each of whichis herein incorporated by reference.

Chimeric Compounds

It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single compound or even at asingle nucleoside within an oligonucleotide.

The present invention also includes antisense compounds which arechimeric compounds. “Chimeric” antisense compounds or “chimeras,” in thecontext of this invention, are antisense compounds, particularlyoligonucleotides, which contain two or more chemically distinct regions,each made up of at least one monomer unit, i.e., a nucleotide in thecase of an oligonucleotide compound. These oligonucleotides typicallycontain at least one region wherein the oligonucleotide is modified soas to confer upon the oligonucleotide increased resistance to nucleasedegradation, increased cellular uptake, increased stability and/orincreased binding affinity for the target nucleic acid. An additionalregion of the oligonucleotide may serve as a substrate for enzymescapable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNAseH is a cellular endonuclease which cleaves the RNA strand of an RNA:DNAduplex. Activation of RNase H, therefore, results in cleavage of the RNAtarget, thereby greatly enhancing the efficiency ofoligonucleotide-mediated inhibition of gene expression. The cleavage ofRNA:RNA hybrids can, in like fashion, be accomplished through theactions of endoribonucleases, such as RNAseL which cleaves both cellularand viral RNA. Cleavage of the RNA target can be routinely detected bygel electrophoresis and, if necessary, associated nucleic acidhybridization techniques known in the art.

Chimeric antisense compounds of the invention may be formed as compositestructures of two or more oligonucleotides, modified oligonucleotides,oligonucleosides and/or oligonucleotide mimetics as described above.Such compounds have also been referred to in the art as hybrids orgapmers. Representative United States patents that teach the preparationof such hybrid structures include, but are not limited to, U.S. Pat.Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711;5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922,certain of which are commonly owned with the instant application, andeach of which is herein incorporated by reference in its entirety.

Salts

The antisense compounds of the invention encompass any pharmaceuticallyacceptable salts, esters, or salts of such esters, or any other compoundwhich, upon administration to an animal, including a human, is capableof providing (directly or indirectly) the biologically active metaboliteor residue thereof. The term “pharmaceutically acceptable salts” refersto physiologically and pharmaceutically acceptable salts of thecompounds of the invention: i.e., salts that retain the desiredbiological activity of the parent compound and do not impart undesiredtoxicological effects thereto. For oligonucleotides, preferred examplesof pharmaceutically acceptable salts and their uses are furtherdescribed in U.S. Pat. No. 6,287,860, which is incorporated herein inits entirety. Sodium salts are especially suitable salts of thecompounds of the present invention.

G. Formulations

The compounds of the invention may also be admixed, encapsulated,conjugated or otherwise associated with other molecules, moleculestructures or mixtures of compounds, as for example, liposomes,receptor-targeted molecules, oral, rectal, topical or otherformulations, for assisting in uptake, distribution and/or absorption.Representative United States patents that teach the preparation of suchuptake, distribution and/or absorption-assisting formulations include,but are not limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016;5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721;4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170;5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854;5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948;5,580,575; and 5,595,756, each of which is herein incorporated byreference.

The present invention also includes pharmaceutical compositions andformulations which include the antisense compounds of the invention. Thepharmaceutical compositions of the present invention may be administeredin a number of ways depending upon whether local or systemic treatmentis desired and upon the area to be treated. Administration may betopical (including ophthalmic and to mucous membranes including vaginaland rectal delivery), pulmonary, e.g., by inhalation or insufflation ofpowders or aerosols, including by nebulizer; intratracheal, intranasal,epidermal and transdermal), oral or parenteral. Parenteraladministration includes intravenous, intraarterial, subcutaneous,intraperitoneal or intramuscular injection or infusion; or intracranial,e.g., intrathecal or intraventricular, administration. Oligonucleotideswith at least one 2′-O-methoxyethyl modification are believed to beparticularly useful for oral administration. Pharmaceutical compositionsand formulations for topical administration may include transdermalpatches, ointments, lotions, creams, gels, drops, suppositories, sprays,liquids and powders. Conventional pharmaceutical carriers, aqueous,powder or oily bases, thickeners and the like may be necessary ordesirable. Coated condoms, gloves and the like may also be useful.

The pharmaceutical formulations of the present invention, which mayconveniently be presented in unit dosage form, may be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present invention may be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, gel capsules, liquid syrups, soft gels, suppositories, andenemas. The compositions of the present invention may also be formulatedas suspensions in aqueous, non-aqueous or mixed media. Aqueoussuspensions may further contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, foams and liposome-containingformulations. The pharmaceutical compositions and formulations of thepresent invention may comprise one or more penetration enhancers,carriers, excipients or other active or inactive ingredients.

Emulsions are typically heterogenous systems of one liquid dispersed inanother in the form of droplets usually exceeding 0.1 μm in diameter.Emulsions may contain additional components in addition to the dispersedphases, and the active drug which may be present as a solution in eitherthe aqueous phase, oily phase or itself as a separate phase.Microemulsions are included as an embodiment of the present invention.Emulsions and their uses are well known in the art and are furtherdescribed in U.S. Pat. No. 6,287,860, which is incorporated herein inits entirety.

Formulations of the present invention include liposomal formulations. Asused in the present invention, the term “liposome” means a vesiclecomposed of amphiphilic lipids arranged in a spherical bilayer orbilayers. Liposomes are unilamellar or multilamellar vesicles which havea membrane formed from a lipophilic material and an aqueous interiorthat contains the composition to be delivered. Cationic liposomes arepositively charged liposomes which are believed to interact withnegatively charged DNA molecules to form a stable complex. Liposomesthat are pH-sensitive or negatively-charged are believed to entrap DNArather than complex with it. Both cationic and noncationic liposomeshave been used to deliver DNA to cells.

Liposomes also include “sterically stabilized” liposomes, a term which,as used herein, refers to liposomes comprising one or more specializedlipids that, when incorporated into liposomes, result in enhancedcirculation lifetimes relative to liposomes lacking such specializedlipids. Examples of sterically stabilized liposomes are those in whichpart of the vesicle-forming lipid portion of the liposome comprises oneor more glycolipids or is derivatized with one or more hydrophilicpolymers, such as a polyethylene glycol (PEG) moiety. Liposomes andtheir uses are further described in U.S. Pat. No. 6,287,860, which isincorporated herein in its entirety.

The pharmaceutical formulations and compositions of the presentinvention may also include surfactants. The use of surfactants in drugproducts, formulations and in emulsions is well known in the art.Surfactants and their uses are further described in U.S. Pat. No.6,287,860, which is incorporated herein in its entirety.

In one embodiment, the present invention employs various penetrationenhancers to effect the efficient delivery of nucleic acids,particularly oligonucleotides. In addition to aiding the diffusion ofnon-lipophilic drugs across cell membranes, penetration enhancers alsoenhance the permeability of lipophilic drugs. Penetration enhancers maybe classified as belonging to one of five broad categories, i.e.,surfactants, fatty acids, bile salts, chelating agents, andnon-chelating non-surfactants. Penetration enhancers and their uses arefurther described in U.S. Pat. No. 6,287,860, which is incorporatedherein in its entirety.

One of skill in the art will recognize that formulations are routinelydesigned according to their intended use, i.e. route of administration.

Preferred formulations for topical administration include those in whichthe oligonucleotides of the invention are in admixture with a topicaldelivery agent such as lipids, liposomes, fatty acids, fatty acidesters, steroids, chelating agents and surfactants. Preferred lipids andliposomes include neutral (e.g. dioleoylphosphatidyl DOPE ethanolamine,dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline)negative (e.g. dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g.dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidylethanolamine DOTMA).

For topical or other administration, oligonucleotides of the inventionmay be encapsulated within liposomes or may form complexes thereto, inparticular to cationic liposomes. Alternatively, oligonucleotides may becomplexed to lipids, in particular to cationic lipids. Preferred fattyacids and esters, pharmaceutically acceptable salts thereof, and theiruses are further described in U.S. Pat. No. 6,287,860, which isincorporated herein in its entirety. Topical formulations are describedin detail in U.S. patent application Ser. No. 09/315,298 filed on May20, 1999, which is incorporated herein by reference in its entirety.

Compositions and formulations for oral administration include powders orgranules, microparticulates, nanoparticulates, suspensions or solutionsin water or non-aqueous media, capsules, gel capsules, sachets, tabletsor minitablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders may be desirable. Preferred oral formulationsare those in which oligonucleotides of the invention are administered inconjunction with one or more penetration enhancers surfactants andchelators. Preferred surfactants include fatty acids and/or esters orsalts thereof, bile acids and/or salts thereof. Preferred bileacids/salts and fatty acids and their uses are further described in U.S.Pat. No. 6,287,860, which is incorporated herein in its entirety. Alsopreferred are combinations of penetration enhancers, for example, fattyacids/salts in combination with bile acids/salts. A particularlypreferred combination is the sodium salt of lauric acid, capric acid andUDCA. Further penetration enhancers include polyoxyethylene-9-laurylether, polyoxyethylene-20-cetyl ether. Oligonucleotides of the inventionmay be delivered orally, in granular form including sprayed driedparticles, or complexed to form micro or nanoparticles. Oligonucleotidecomplexing agents and their uses are further described in U.S. Pat. No.6,287,860, which is incorporated herein in its entirety. Oralformulations for oligonucleotides and their preparation are described indetail in U.S. application Ser. Nos. 09/108,673 (filed Jul. 1, 1998),09/315,298 (filed May 20, 1999) and 10/071,822, filed Feb. 8, 2002, eachof which is incorporated herein by reference in their entirety.

Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionswhich may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

Certain embodiments of the invention provide pharmaceutical compositionscontaining one or more oligomeric compounds and one or more otherpharmaceutical agents which function by a non-antisense mechanism.Examples of such pharmaceutical agents include but are not limited tocancer chemotherapeutic drugs, anti-inflammatory drugs, anti-viraldrugs, and compounds for treatment of metabolic diseases such asdiabetes, high blood sugar or obesity, or cardiovascular conditions suchas elevated blood cholesterol or blood pressure. Combinations ofantisense compounds and other non-antisense drugs are also within thescope of this invention. Two or more combined compounds may be usedtogether or sequentially. When used with the compounds of the invention,such pharmaceutical agents may be used individually (e.g., rosiglitazoneand oligonucleotide), sequentially (e.g., 5-fluorouracil andoligonucleotide for a period of time followed by methotrexate andoligonucleotide), or in combination with one or more other treatments(e.g., 5-fluorouracil, methotrexate and oligonucleotide, or5-fluorouracil, radiotherapy and oligonucleotide).

In another related embodiment, compositions of the invention may containone or more antisense compounds, particularly oligonucleotides, targetedto a first nucleic acid and one or more additional antisense compoundstargeted to a second nucleic acid target. Alternatively, compositions ofthe invention may contain two or more antisense compounds targeted todifferent regions of the same nucleic acid target. Numerous examples ofantisense compounds are known in the art. Two or more combined compoundsmay be used together or sequentially.

H. Dosing

The formulation of therapeutic compositions and their subsequentadministration (dosing) is believed to be within the skill of those inthe art. Dosing is dependent on severity and responsiveness of thedisease state to be treated, with the course of treatment lasting fromseveral days to several months, or until a cure is effected or adiminution of the disease state is achieved. Optimal dosing schedulescan be calculated from measurements of drug accumulation in the body ofthe patient. Persons of ordinary skill can easily determine optimumdosages, dosing methodologies and repetition rates. Optimum dosages mayvary depending on the relative potency of individual oligonucleotides,and can generally be estimated based on EC₅₀s found to be effective inin vitro and in vivo animal models. In general, dosage is from 0.01 ugto 100 g per kg of body weight, and may be given once or more daily,weekly, monthly or yearly, or even once every 2 to 20 years. Persons ofordinary skill in the art can easily estimate repetition rates fordosing based on measured residence times and concentrations of the drugin bodily fluids or tissues. Following successful treatment, it may bedesirable to have the patient undergo maintenance therapy to prevent therecurrence of the disease state, wherein the oligonucleotide isadministered in maintenance doses, ranging from 0.01 ug to 100 g per kgof body weight, once or more daily, to once every 20 years.

While the present invention has been described with specificity inaccordance with certain of its preferred embodiments, the followingexamples serve only to illustrate the invention and are not intended tolimit the same.

EXAMPLES Example 1 Synthesis of Nucleoside Phosphoramidites

The following compounds, including amidites and their intermediates wereprepared as described in U.S. Pat. No. 6,426,220 and published PCT WO02/36743; 5′-O-Dimethoxytrityl-thymidine intermediate for 5-methyl dCamidite, 5′-O-Dimethoxytrityl-2′-deoxy-5-methylcytidine intermediate for5-methyl-dC amidite,5′-O-Dimethoxytrityl-2′-deoxy-N4-benzoyl-5-methylcytidine penultimateintermediate for 5-methyl dC amidite,[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-deoxy-N⁴-benzoyl-5-methylcytidin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(5-methyl dC amidite), 2′-Fluorodeoxyadenosine, 2′-Fluorodeoxyguanosine,2′-Fluorouridine, 2′-Fluorodeoxycytidine, 2′-O-(2-Methoxyethyl) modifiedamidites, 2′-O-(2-methoxyethyl)-5-methyluridine intermediate,5′-O-DMT-2′-O-(2-methoxyethyl)-5-methyluridine penultimate intermediate,[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-5-methyluridin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE T amidite),5′-O-Dimethoxytrityl-2′-O-(2-methoxyethyl)-5-methylcytidineintermediate,5′-O-dimethoxytrityl-2′-O-(2-methoxyethyl)-N⁴-benzoyl-5-methyl-cytidinepenultimate intermediate,[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-benzoyl-5-methylcytidin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE 5-Me-C amidite),[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁶-benzoyladenosin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE A amdite),[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-isobutyrylguanosin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE G amidite), 2′-O-(Aminooxyethyl) nucleoside amidites and2′-O-(dimethylaminooxyethyl) nucleoside amidites,2′-(Dimethylaminooxyethoxy) nucleoside amidites,5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine,5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine,2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine,5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine,5′-O-tert-Butyldiphenylsilyl-2′-O—[N,Ndimethylaminooxyethyl]-5-methyluridine,2′-O-(dimethylaminooxyethyl)-5-methyluridine,5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine,5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite],2′-(Aminooxyethoxy) nucleoside amidites,N2-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite],2′-dimethylaminoethoxyethoxy (2′-DMAEOE) nucleoside amidites,2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl uridine,5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyluridine and5′-O-Dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyluridine-3′-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite.

Example 2 Oligonucleotide and Oligonucleoside Synthesis

The antisense compounds used in accordance with this invention may beconveniently and routinely made through the well-known technique ofsolid phase synthesis. Equipment for such synthesis is sold by severalvendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is well known to usesimilar techniques to prepare oligonucleotides such as thephosphorothioates and alkylated derivatives.

Oligonucleotides: Unsubstituted and substituted phosphodiester (P=0)oligonucleotides are synthesized on an automated DNA synthesizer(Applied Biosystems model 394) using standard phosphoramidite chemistrywith oxidation by iodine.

Phosphorothioates (P═S) are synthesized similar to phosphodiesteroligonucleotides with the following exceptions: thiation was effected byutilizing a 10% w/v solution of 3,H-1,2-benzodithiole-3-one 1,1-dioxidein acetonitrile for the oxidation of the phosphite linkages. Thethiation reaction step time was increased to 180 sec and preceded by thenormal capping step. After cleavage from the CPG column and deblockingin concentrated ammonium hydroxide at 55° C. (12-16 hr), theoligonucleotides were recovered by precipitating with >3 volumes ofethanol from a 1 M NH₄OAc solution. Phosphinate oligonucleotides areprepared as described in U.S. Pat. No. 5,508,270, herein incorporated byreference.

Alkyl phosphonate oligonucleotides are prepared as described in U.S.Pat. No. 4,469,863, herein incorporated by reference.

3′-Deoxy-3′-methylene phosphonate oligonucleotides are prepared asdescribed in U.S. Pat. No. 5,610,289 or 5,625,050, herein incorporatedby reference.

Phosphoramidite oligonucleotides are prepared as described in U.S. Pat.No. 5,256,775 or U.S. Pat. No. 5,366,878, herein incorporated byreference.

Alkylphosphonothioate oligonucleotides are prepared as described inpublished PCT applications PCT/US94/00902 and PCT/US93/06976 (publishedas WO 94/17093 and WO 94/02499, respectively), herein incorporated byreference.

3′-Deoxy-3′-amino phosphoramidate oligonucleotides are prepared asdescribed in U.S. Pat. No. 5,476,925, herein incorporated by reference.

Phosphotriester oligonucleotides are prepared as described in U.S. Pat.No. 5,023,243, herein incorporated by reference.

Borano phosphate oligonucleotides are prepared as described in U.S. Pat.Nos. 5,130,302 and 5,177,198, both herein incorporated by reference.

Oligonucleosides: Methylenemethylimino linked oligonucleosides, alsoidentified as MMI linked oligonucleosides, methylenedimethylhydrazolinked oligonucleosides, also identified as MDH linked oligonucleosides,and methylenecarbonylamino linked oligonucleosides, also identified asamide-3 linked oligonucleosides, and methyleneaminocarbonyl linkedoligonucleosides, also identified as amide-4 linked oligonucleosides, aswell as mixed backbone compounds having, for instance, alternating MMIand P═O or P═S linkages are prepared as described in U.S. Pat. Nos.5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289, all of whichare herein incorporated by reference.

Formacetal and thioformacetal linked oligonucleosides are prepared asdescribed in U.S. Pat. Nos. 5,264,562 and 5,264,564, herein incorporatedby reference.

Ethylene oxide linked oligonucleosides are prepared as described in U.S.Pat. No. 5,223,618, herein incorporated by reference.

Example 3 RNA Synthesis

In general, RNA synthesis chemistry is based on the selectiveincorporation of various protecting groups at strategic intermediaryreactions. Although one of ordinary skill in the art will understand theuse of protecting groups in organic synthesis, a useful class ofprotecting groups includes silyl ethers. In particular bulky silylethers are used to protect the 5′-hydroxyl in combination with anacid-labile orthoester protecting group on the 2′-hydroxyl. This set ofprotecting groups is then used with standard solid-phase synthesistechnology. It is important to lastly remove the acid labile orthoesterprotecting group after all other synthetic steps. Moreover, the earlyuse of the silyl protecting groups during synthesis ensures facileremoval when desired, without undesired deprotection of 2′ hydroxyl.

Following this procedure for the sequential protection of the5′-hydroxyl in combination with protection of the 2′-hydroxyl byprotecting groups that are differentially removed and are differentiallychemically labile, RNA oligonucleotides were synthesized.

RNA oligonucleotides are synthesized in a stepwise fashion. Eachnucleotide is added sequentially (3′- to 5′-direction) to a solidsupport-bound oligonucleotide. The first nucleoside at the 3′-end of thechain is covalently attached to a solid support. The nucleotideprecursor, a ribonucleoside phosphoramidite, and activator are added,coupling the second base onto the 5′-end of the first nucleoside. Thesupport is washed and any unreacted 5′-hydroxyl groups are capped withacetic anhydride to yield 5′-acetyl moieties. The linkage is thenoxidized to the more stable and ultimately desired P(V) linkage. At theend of the nucleotide addition cycle, the 5′-silyl group is cleaved withfluoride. The cycle is repeated for each subsequent nucleotide.

Following synthesis, the methyl protecting groups on the phosphates arecleaved in 30 minutes utilizing 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate (S₂Na₂)in DMF. The deprotection solution is washed from the solid support-boundoligonucleotide using water. The support is then treated with 40%methylamine in water for 10 minutes at 55° C. This releases the RNAoligonucleotides into solution, deprotects the exocyclic amines, andmodifies the 2′-groups. The oligonucleotides can be analyzed by anionexchange HPLC at this stage.

The 2′-orthoester groups are the last protecting groups to be removed.The ethylene glycol monoacetate orthoester protecting group developed byDharmacon Research, Inc. (Lafayette, Colo.), is one example of a usefulorthoester protecting group which, has the following importantproperties. It is stable to the conditions of nucleoside phosphoramiditesynthesis and oligonucleotide synthesis. However, after oligonucleotidesynthesis the oligonucleotide is treated with methylamine which not onlycleaves the oligonucleotide from the solid support but also removes theacetyl groups from the orthoesters. The resulting 2-ethyl-hydroxylsubstituents on the orthoester are less electron withdrawing than theacetylated precursor. As a result, the modified orthoester becomes morelabile to acid-catalyzed hydrolysis. Specifically, the rate of cleavageis approximately 10 times faster after the acetyl groups are removed.Therefore, this orthoester possesses sufficient stability in order to becompatible with oligonucleotide synthesis and yet, when subsequentlymodified, permits deprotection to be carried out under relatively mildaqueous conditions compatible with the final RNA oligonucleotideproduct.

Additionally, methods of RNA synthesis are well known in the art(Scaringe, S. A. Ph.D. Thesis, University of Colorado, 1996; Scaringe,S. A., et al., J. Am. Chem. Soc., 1998, 120, 11820-11821; Matteucci, M.D. and Caruthers, M. H. J. Am. Chem. Soc., 1981, 103, 3185-3191;Beaucage, S. L. and Caruthers, M. H. Tetrahedron Lett., 1981, 22,1859-1862; Dahl, B. J., et al., Acta Chem. Scand. 1990, 44, 639-641;Reddy, M. P., et al., Tetrahedrom Lett., 1994, 25, 4311-4314; Wincott,F. et al., Nucleic Acids Res., 1995, 23, 2677-2684; Griffin, B. E., etal., Tetrahedron, 1967, 23, 2301-2313; Griffin, B. E., et al.,Tetrahedron, 1967, 23, 2315-2331).

RNA antisense compounds (RNA oligonucleotides) of the present inventioncan be synthesized by the methods herein or purchased from DharmaconResearch, Inc (Lafayette, Colo.). Once synthesized, complementary RNAantisense compounds can then be annealed by methods known in the art toform double stranded (duplexed) antisense compounds. For example,duplexes can be formed by combining 30 μl of each of the complementarystrands of RNA oligonucleotides (50 uM RNA oligonucleotide solution) and15 μl of 5× annealing buffer (100 mM potassium acetate, 30 mM HEPES-KOHpH 7.4, 2 mM magnesium acetate) followed by heating for 1 minute at 90°C., then 1 hour at 37° C. The resulting duplexed antisense compounds canbe used in kits, assays, screens, or other methods to investigate therole of a target nucleic acid.

Example 4 Synthesis of Chimeric Oligonucleotides

Chimeric oligonucleotides, oligonucleosides or mixedoligonucleotides/oligonucleosides of the invention can be of severaldifferent types. These include a first type wherein the “gap” segment oflinked nucleosides is positioned between 5′ and 3′ “wing” segments oflinked nucleosides and a second “open end” type wherein the “gap”segment is located at either the 3′ or the 5′ terminus of the oligomericcompound. Oligonucleotides of the first type are also known in the artas “gapmers” or gapped oligonucleotides. Oligonucleotides of the secondtype are also known in the art as “hemimers” or “wingmers”.

[2′-O-Me]-[2′-deoxy]-[2′-O-Me]Chimeric Phosphorothioate Oligonucleotides

Chimeric oligonucleotides having 2′-O-alkyl phosphorothioate and2′-deoxy phosphorothioate oligonucleotide segments are synthesized usingan Applied Biosystems automated DNA synthesizer Model 394, as above.Oligonucleotides are synthesized using the automated synthesizer and2′-deoxy-5′-dimethoxytrityl-3′-O-phosphoramidite for the DNA portion and5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite for 5′ and 3′ wings.The standard synthesis cycle is modified by incorporating coupling stepswith increased reaction times for the5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite. The fully protectedoligonucleotide is cleaved from the support and deprotected inconcentrated ammonia (NH₄OH) for 12-16 hr at 55° C. The deprotectedoligo is then recovered by an appropriate method (precipitation, columnchromatography, volume reduced in vacuo and analyzedspetrophotometrically for yield and for purity by capillaryelectrophoresis and by mass spectrometry.

[2′-O-(2-Methoxyethyl)]-[2′-deoxy]-[2′-O-(Methoxyethyl)]ChimericPhosphorothioate Oligonucleotides

[2′-O-(2-methoxyethyl)]-[2′-deoxy]-[-2′-O-(methoxyethyl)]chimericphosphorothioate oligonucleotides were prepared as per the procedureabove for the 2′-O-methyl chimeric oligonucleotide, with thesubstitution of 2′-O-(methoxyethyl)amidites for the 2′-O-methylamidites.

[2′-O-(2-Methoxyethyl)Phosphodiester]-[2′-deoxyPhosphorothioate]-[2′-O-(2-Methoxyethyl)Phosphodiester]ChimericOligonucleotides

[2′-O-(2-methoxyethyl phosphodiester]-[2′-deoxyphosphorothioate]-[2′-O-(methoxyethyl)phosphodiester]chimericoligonucleotides are prepared as per the above procedure for the2′-O-methyl chimeric oligonucleotide with the substitution of2′-O-(methoxyethyl)amidites for the 2′-O-methyl amidites, oxidation withiodine to generate the phosphodiester internucleotide linkages withinthe wing portions of the chimeric structures and sulfurization utilizing3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) to generatethe phosphorothioate internucleotide linkages for the center gap.

Other chimeric oligonucleotides, chimeric oligonucleosides and mixedchimeric oligonucleotides/oligonucleosides are synthesized according toU.S. Pat. No. 5,623,065, herein incorporated by reference.

Example 5 Design and Screening of Duplexed Antisense Compounds TargetingGlucagon Receptor

In accordance with the present invention, a series of nucleic acidduplexes comprising the antisense compounds of the present invention andtheir complements can be designed to target glucagon receptor. Thenucleobase sequence of the antisense strand of the duplex comprises atleast an 8-nucleobase portion of an oligonucleotide in Table 1. The endsof the strands may be modified by the addition of one or more natural ormodified nucleobases to form an overhang. The sense strand of the dsRNAis then designed and synthesized as the complement of the antisensestrand and may also contain modifications or additions to eitherterminus. For example, in one embodiment, both strands of the dsRNAduplex would be complementary over the central nucleobases, each havingoverhangs at one or both termini.

For example, a duplex comprising an antisense strand having the sequenceCGAGAGGCGGACGGGACCG (SEQ ID NO.: 824) and having a two-nucleobaseoverhang of deoxythymidine (dT) would have the following structure:

In another embodiment, a duplex comprising an antisense strand havingthe same sequence CGAGAGGCGGACGGGACCG (SEQ ID NO: 824) may be preparedwith blunt ends (no single strand overhang) as shown:

The RNA duplex can be unimolecular or biomolecular; i.e., the twostrands can be part of a single molecule or may be separate molecules.RNA strands of the duplex can be synthesized by methods disclosed hereinor purchased from Dharmacon Research Inc., (Lafayette, Colo.). Oncesynthesized, the complementary strands are annealed. The single strandsare aliquoted and diluted to a concentration of 50 uM. Once diluted, 30uL of each strand is combined with 15 uL of a 5× solution of annealingbuffer. The final concentration of said buffer is 100 mM potassiumacetate, 30 mM HEPES-KOH pH 7.4, and 2 mM magnesium acetate. The finalvolume is 75 uL. This solution is incubated for 1 minute at 90° C. andthen centrifuged for 15 seconds. The tube is allowed to sit for 1 hourat 37° C. at which time the dsRNA duplexes are used in experimentation.The final concentration of the dsRNA duplex is 20 uM. This solution canbe stored frozen (−20° C.) and freeze-thawed up to 5 times.

Once prepared, the duplexed antisense compounds are evaluated for theirability to modulate glucagon receptor expression.

When cells reached 80% confluency, they are treated with duplexedantisense compounds of the invention. For cells grown in 96-well plates,wells are washed once with 200 μL OPTI-MEM-1 reduced-serum medium (GibcoBRL) and then treated with 130 μL of OPTI-MEM-1 containing 12 μg/mLLIPOFECTIN (Gibco BRL) and the desired duplex antisense compound at afinal concentration of 200 nM. After 5 hours of treatment, the medium isreplaced with fresh medium. Cells are harvested 16 hours aftertreatment, at which time RNA is isolated and target reduction measuredby RT-PCR.

Example 6 Oligonucleotide Isolation

After cleavage from the controlled pore glass solid support anddeblocking in concentrated ammonium hydroxide at 55° C. for 12-16 hours,the oligonucleotides or oligonucleosides are recovered by precipitationout of 1 M NH₄OAc with >3 volumes of ethanol. Synthesizedoligonucleotides are analyzed by electrospray mass spectroscopy(molecular weight determination) and by capillary gel electrophoresisand judged to be at least 70% full length material. The relative amountsof phosphorothioate and phosphodiester linkages obtained in thesynthesis is determined by the ratio of correct molecular weightrelative to the −16 amu product (+/−32+/−48). For some studiesoligonucleotides are purified by HPLC, as described by Chiang et al., J.Biol. Chem. 1991, 266, 18162-18171. Results obtained with HPLC-purifiedmaterial are similar to those obtained with non-HPLC purified material.

Example 7 Oligonucleotide Synthesis 96 Well Plate Format

Oligonucleotides are synthesized via solid phase P(III) phosphoramiditechemistry on an automated synthesizer capable of assembling 96 sequencessimultaneously in a 96-well format. Phosphodiester internucleotidelinkages are afforded by oxidation with aqueous iodine. Phosphorothioateinternucleotide linkages are generated by sulfurization utilizing3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) in anhydrousacetonitrile. Standard base-protected beta-cyanoethyl-diiso-propylphosphoramidites are purchased from commercial vendors (e.g. PE-AppliedBiosystems, Foster City, Calif., or Pharmacia, Piscataway, N.J.).Non-standard nucleosides are synthesized as per standard or patentedmethods. They are utilized as base protected beta-cyanoethyldiisopropylphosphoramidites.

Oligonucleotides are cleaved from support and deprotected withconcentrated NH₄OH at elevated temperature (55-60° C.) for 12-16 hoursand the released product then dried in vacuo. The dried product is thenre-suspended in sterile water to afford a master plate from which allanalytical and test plate samples are then diluted utilizing roboticpipettors.

Example 8 Oligonucleotide Analysis 96-Well Plate Format

The concentration of oligonucleotide in each well is assessed bydilution of samples and UV absorption spectroscopy. The full-lengthintegrity of the individual products is evaluated by capillaryelectrophoresis (CE) in either the 96-well format (Beckman P/ACE™ MDQ)or, for individually prepared samples, on a commercial CE apparatus(e.g., Beckman P/ACE™ 5000, ABI 270). Base and backbone composition isconfirmed by mass analysis of the compounds utilizing electrospray-massspectroscopy. All assay test plates are diluted from the master plateusing single and multi-channel robotic pipettors. Plates are judged tobe acceptable if at least 85% of the compounds on the plate are at least85% full length.

Example 9 Cell Culture and Oligonucleotide Treatment

The effect of antisense compounds on target nucleic acid expression canbe tested in any of a variety of cell types provided that the targetnucleic acid is present at measurable levels. This can be routinelydetermined using, for example, PCR or Northern blot analysis. Thefollowing cell types are provided for illustrative purposes, but othercell types can be routinely used, provided that the target is expressedin the cell type chosen. This can be readily determined by methodsroutine in the art, for example Northern blot analysis, ribonucleaseprotection assays, or RT-PCR.

T-24 Cells:

The human transitional cell bladder carcinoma cell line T-24 is obtainedfrom the American Type Culture Collection (ATCC) (Manassas, Va.). T-24cells are routinely cultured in complete McCoy's 5A basal media(Invitrogen Corporation, Carlsbad, Calif.) supplemented with 10% fetalcalf serum (Invitrogen Corporation, Carlsbad, Calif.), penicillin 100units per mL, and streptomycin 100 micrograms per mL (InvitrogenCorporation, Carlsbad, Calif.). Cells are routinely passaged bytrypsinization and dilution when they reached 90% confluence. Cells areseeded into 96-well plates (Falcon-Primaria #353872) at a density of7000 cells/well for use in RT-PCR analysis.

For Northern blotting or other analysis, cells may be seeded onto 100 mmor other standard tissue culture plates and treated similarly, usingappropriate volumes of medium and oligonucleotide.

A549 Cells:

The human lung carcinoma cell line A549 is obtained from the AmericanType Culture Collection (ATCC) (Manassas, Va.). A549 cells are routinelycultured in DMEM basal media (Invitrogen Corporation, Carlsbad, Calif.)supplemented with 10% fetal calf serum (Invitrogen Corporation,Carlsbad, Calif.), penicillin 100 units per mL, and streptomycin 100micrograms per mL (Invitrogen Corporation, Carlsbad, Calif.). Cells areroutinely passaged by trypsinization and dilution when they reach 90%confluence.

NHDF Cells:

Human neonatal dermal fibroblast (NHDF) are obtained from the CloneticsCorporation (Walkersville, Md.). NHDFs are routinely maintained inFibroblast Growth Medium (Clonetics Corporation, Walkersville, Md.)supplemented as recommended by the supplier. Cells are maintained for upto 10 passages as recommended by the supplier.

HEK Cells:

Human embryonic keratinocytes (HEK) are obtained from the CloneticsCorporation (Walkersville, Md.). HEKs are routinely maintained inKeratinocyte Growth Medium (Clonetics Corporation, Walkersville, Md.)formulated as recommended by the supplier. Cells are routinelymaintained for up to 10 passages as recommended by the supplier.

HepG2 Cells:

The human hepatoblastoma cell line HepG2 is obtained from the AmericanType Culture Collection (Manassas, Va.). HepG2 cells are routinelycultured in Eagle's MEM supplemented with 10% fetal calf serum,non-essential amino acids, and 1 mM sodium pyruvate (Gibco/LifeTechnologies, Gaithersburg, Md.). Cells are routinely passaged bytrypsinization and dilution when they reach 90% confluence. Cells areseeded into 96-well plates (Falcon-Primaria #3872) at a density of 7000cells/well for use in RT-PCR analysis.

For Northern blotting or other analyses, cells may be seeded onto 100 mmor other standard tissue culture plates and treated similarly, usingappropriate volumes of medium and oligonucleotide.

Primary Mouse Hepatocytes

Primary mouse hepatocytes are prepared from CD-1 mice purchased fromCharles River Labs. Primary mouse hepatocytes are routinely cultured inHepatocyte Attachment Media (Gibco) supplemented with 10% Fetal BovineSerum (Gibco/Life Technologies, Gaithersburg, Md.), 250 nM dexamethasone(Sigma), 10 nM bovine insulin (Sigma). Cells are seeded into 96-wellplates (Falcon-Primaria #3872) at a density of 10000 cells/well for usein RT-PCR analysis.

For Northern blotting or other analyses, cells may be seeded onto 100 mmor other standard tissue culture plates and treated similarly, usingappropriate volumes of medium and oligonucleotide.

Treatment with Antisense Compounds:

When cells reached 65-75% confluency, they are treated witholigonucleotide. For cells grown in 96-well plates, wells are washedonce with 100 μL OPTI-MEM™-1 reduced-serum medium (InvitrogenCorporation, Carlsbad, Calif.) and then treated with 130 μL ofOPTI-MEM™-1 containing 3.75 μg/mL LIPOFECTIN™ (Invitrogen Corporation,Carlsbad, Calif.) and the desired concentration of oligonucleotide.Cells are treated and data are obtained in triplicate. After 4-7 hoursof treatment at 37° C., the medium is replaced with fresh medium. Cellsare harvested 16-24 hours after oligonucleotide treatment.

The concentration of oligonucleotide used varies from cell line to cellline. To determine the optimal oligonucleotide concentration for aparticular cell line, the cells are treated with a positive controloligonucleotide at a range of concentrations. For human cells thepositive control oligonucleotide is selected from either ISIS 13920(TCCGTCATCGCTCCTCAGGG, SEQ ID NO: 1) which is targeted to human H-ras,or ISIS 18078, (GTGCGCGCGAGCCCGAAATC, SEQ ID NO: 2) which is targeted tohuman Jun-N-terminal kinase-2 (JNK2). Both controls are2′-O-methoxyethyl gapmers (2′-O-methoxyethyls shown in bold) with aphosphorothioate backbone. For mouse or rat cells the positive controloligonucleotide is ISIS 15770, ATGCATTCTGCCCCCAAGGA, SEQ ID NO: 3, a2′-O-methoxyethyl gapmer (2′-O-methoxyethyls shown in bold) with aphosphorothioate backbone which is targeted to both mouse and rat c-raf.The concentration of positive control oligonucleotide that results in80% inhibition of c-H-ras (for ISIS 13920), JNK2 (for ISIS 18078) orc-raf (for ISIS 15770) mRNA is then utilized as the screeningconcentration for new oligonucleotides in subsequent experiments forthat cell line. If 80% inhibition is not achieved, the lowestconcentration of positive control oligonucleotide that results in 60%inhibition of c-H-ras, JNK2 or c-raf mRNA is then utilized as theoligonucleotide screening concentration in subsequent experiments forthat cell line. If 60% inhibition is not achieved, that particular cellline is deemed as unsuitable for oligonucleotide transfectionexperiments. The concentrations of antisense oligonucleotides usedherein are from 50 nM to 300 nM.

Example 10 Analysis of Oligonucleotide Inhibition of Glucagon ReceptorExpression

Antisense modulation of glucagon receptor expression can be assayed in avariety of ways known in the art. For example, glucagon receptor mRNAlevels can be quantitated by, e.g., Northern blot analysis, competitivepolymerase chain reaction (PCR), or real-time PCR (RT-PCR). Real-timequantitative PCR is presently preferred. RNA analysis can be performedon total cellular RNA or poly(A)+ mRNA. The preferred method of RNAanalysis of the present invention is the use of total cellular RNA asdescribed in other examples herein. Methods of RNA isolation are wellknown in the art. Northern blot analysis is also routine in the art.Real-time quantitative (PCR) can be conveniently accomplished using thecommercially available ABI PRISM™ 7600, 7700, or 7900 Sequence DetectionSystem, available from PE-Applied Biosystems, Foster City, Calif. andused according to manufacturer's instructions.

Protein levels of glucagon receptor can be quantitated in a variety ofways well known in the art, such as immunoprecipitation, Western blotanalysis (immunoblotting), enzyme-linked immunosorbent assay (ELISA) orfluorescence-activated cell sorting (FACS). Antibodies directed toglucagon receptor can be identified and obtained from a variety ofsources, such as the MSRS catalog of antibodies (Aerie Corporation,Birmingham, Mich.), or can be prepared via conventional monoclonal orpolyclonal antibody generation methods well known in the art.

Example 11 Design of Phenotypic Assays and In Vivo Studies for the Useof Glucagon Receptor Inhibitors Phenotypic Assays

Once glucagon receptor inhibitors have been identified by the methodsdisclosed herein, the compounds are further investigated in one or morephenotypic assays, each having measurable endpoints predictive ofefficacy in the treatment of a particular disease state or condition.Phenotypic assays, kits and reagents for their use are well known tothose skilled in the art and are herein used to investigate the roleand/or association of glucagon receptor in health and disease.Representative phenotypic assays, which can be purchased from any one ofseveral commercial vendors, include those for determining cellviability, cytotoxicity, proliferation or cell survival (MolecularProbes, Eugene, Oreg.; PerkinElmer, Boston, Mass.), protein-based assaysincluding enzymatic assays (Panvera, LLC, Madison, Wis.; BD Biosciences,Franklin Lakes, N.J.; Oncogene Research Products, San Diego, Calif.),cell regulation, signal transduction, inflammation, oxidative processesand apoptosis (Assay Designs Inc., Ann Arbor, Mich.), triglycerideaccumulation (Sigma-Aldrich, St. Louis, Mo.), angiogenesis assays, tubeformation assays, cytokine and hormone assays and metabolic assays(Chemicon International Inc., Temecula, Calif.; Amersham Biosciences,Piscataway, N.J.).

In one non-limiting example, cells determined to be appropriate for aparticular phenotypic assay (i.e., MCF-7 cells selected for breastcancer studies; adipocytes for obesity studies) are treated withglucagon receptor inhibitors identified from the in vitro studies aswell as control compounds at optimal concentrations which are determinedby the methods described above. At the end of the treatment period,treated and untreated cells are analyzed by one or more methods specificfor the assay to determine phenotypic outcomes and endpoints.

Phenotypic endpoints include changes in cell morphology over time ortreatment dose as well as changes in levels of cellular components suchas proteins, lipids, nucleic acids, hormones, saccharides or metals.Measurements of cellular status which include pH, stage of the cellcycle, intake or excretion of biological indicators by the cell, arealso endpoints of interest.

Analysis of the geneotype of the cell (measurement of the expression ofone or more of the genes of the cell) after treatment is also used as anindicator of the efficacy or potency of the glucagon receptorinhibitors. Hallmark genes, or those genes suspected to be associatedwith a specific disease state, condition, or phenotype, are measured inboth treated and untreated cells.

In Vivo Studies

The individual subjects of the in vivo studies described herein arewarm-blooded vertebrate animals, which includes humans.

The clinical trial is subjected to rigorous controls to ensure thatindividuals are not unnecessarily put at risk and that they are fullyinformed about their role in the study. To account for the psychologicaleffects of receiving treatments, volunteers are randomly given placeboor glucagon receptor inhibitor. Furthermore, to prevent the doctors frombeing biased in treatments, they may not be informed as to whether themedication they are administering is a glucagon receptor inhibitor or aplacebo. Using this randomization approach, each volunteer has the samechance of being given either the new treatment or the placebo.

Volunteers may receive either the glucagon receptor inhibitor or placebofor eight week period with biological parameters associated with theindicated disease state or condition being measured at the beginning(baseline measurements before any treatment), end (after the finaltreatment), and at regular intervals during the study period. Suchmeasurements may include the levels of nucleic acid molecules encodingglucagon receptor or glucagon receptor protein levels in body fluids,tissues or organs compared to pre-treatment levels. Other measurementsmay include, but are not limited to, indices of the disease state orcondition being treated, body weight, blood pressure, serum titers ofpharmacologic indicators of disease or toxicity as well as ADME(absorption, distribution, metabolism and excretion) measurements.

Information recorded for each patient may include age (years), gender,height (cm), family history of disease state or condition (yes/no),motivation rating (some/moderate/great) and number and type of previoustreatment regimens for the indicated disease or condition.

Volunteers taking part in this study are healthy adults (age 18 to 65years) and, typically, roughly an equal number of males and femalesparticipate in the study. Volunteers with certain characteristics areequally distributed for placebo and glucagon receptor inhibitortreatment. In general, the volunteers treated with placebo have littleor no response to treatment, whereas the volunteers treated with theglucagon receptor inhibitor show positive trends in their disease stateor condition index at the conclusion of the study.

One of ordinary skill will know how to conduct an appropriate clinicaltrial and will recognize that this is just one of many protocols whichmay be appropriately used.

Example 12 RNA Isolation

Poly(A)+ mRNA Isolation

Poly(A)+ mRNA was isolated according to Miura et al., (Clin. Chem.,1996, 42, 1758-1764). Other methods for poly(A)+ mRNA isolation areroutine in the art. Briefly, for cells grown on 96-well plates, growthmedium was removed from the cells and each well was washed with 200 μLcold PBS. 60 μL lysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 MNaCl, 0.5% NP-40, 20 mM vanadyl-ribonucleoside complex) was added toeach well, the plate was gently agitated and then incubated at roomtemperature for five minutes. 55 μL of lysate was transferred to Oligod(T) coated 96-well plates (AGCT Inc., Irvine Calif.). Plates wereincubated for 60 minutes at room temperature, washed 3 times with 200 μLof wash buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After thefinal wash, the plate was blotted on paper towels to remove excess washbuffer and then air-dried for 5 minutes. 60 μL of elution buffer (5 mMTris-HCl pH 7.6), preheated to 70° C., was added to each well, the platewas incubated on a 90° C. hot plate for 5 minutes, and the eluate wasthen transferred to a fresh 96-well plate.

Cells grown on 100 mm or other standard plates may be treated similarly,using appropriate volumes of all solutions.

Total RNA Isolation

Total RNA was isolated using an RNEASY 96™ kit and buffers purchasedfrom Qiagen Inc. (Valencia, Calif.) following the manufacturer'srecommended procedures. Briefly, for cells grown on 96-well plates,growth medium was removed from the cells and each well was washed with200 μL cold PBS. 150 μL Buffer RLT was added to each well and the platevigorously agitated for 20 seconds. 150 μL of 70% ethanol was then addedto each well and the contents mixed by pipetting three times up anddown. The samples were then transferred to the RNEASY 96™ well plateattached to a QIAVAC™ manifold fitted with a waste collection tray andattached to a vacuum source. Vacuum was applied for 1 minute. 500 μl, ofBuffer RW1 was added to each well of the RNEASY 96™ plate and incubatedfor 15 minutes and the vacuum was again applied for 1 minute. Anadditional 500 μL of Buffer RW1 was added to each well of the RNEASY 96™plate and the vacuum was applied for 2 minutes. 1 mL of Buffer RPE wasthen added to each well of the RNEASY 96™ plate and the vacuum appliedfor a period of 90 seconds. The Buffer RPE wash was then repeated andthe vacuum was applied for an additional 3 minutes. The plate was thenremoved from the QIAVAC™ manifold and blotted dry on paper towels. Theplate was then re-attached to the QIAVAC™ manifold fitted with acollection tube rack containing 1.2 mL collection tubes. RNA was theneluted by pipetting 1404 of RNAse free water into each well, incubating1 minute, and then applying the vacuum for 3 minutes.

The repetitive pipetting and elution steps may be automated using aQIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia Calif.). Essentially,after lysing of the cells on the culture plate, the plate is transferredto the robot deck where the pipetting, DNase treatment and elution stepsare carried out.

Example 13 Real-Time Quantitative PCR Analysis of Glucagon Receptor mRNALevels

Quantitation of glucagon receptor mRNA levels was accomplished byreal-time quantitative PCR using the ABI PRISM™ 7600, 7700, or 7900Sequence Detection System (PE-Applied Biosystems, Foster City, Calif.)according to manufacturer's instructions. This is a closed-tube,non-gel-based, fluorescence detection system which allowshigh-throughput quantitation of polymerase chain reaction (PCR) productsin real-time. As opposed to standard PCR in which amplification productsare quantitated after the PCR is completed, products in real-timequantitative PCR are quantitated as they accumulate. This isaccomplished by including in the PCR reaction an oligonucleotide probethat anneals specifically between the forward and reverse PCR primers,and contains two fluorescent dyes. A reporter dye (e.g., FAM or JOE,obtained from either PE-Applied Biosystems, Foster City, Calif., OperonTechnologies Inc., Alameda, Calif. or Integrated DNA Technologies Inc.,Coralville, Iowa) is attached to the 5′ end of the probe and a quencherdye (e.g., TAMRA, obtained from either PE-Applied Biosystems, FosterCity, Calif., Operon Technologies Inc., Alameda, Calif. or IntegratedDNA Technologies Inc., Coralville, Iowa) is attached to the 3′ end ofthe probe. When the probe and dyes are intact, reporter dye emission isquenched by the proximity of the 3′ quencher dye. During amplification,annealing of the probe to the target sequence creates a substrate thatcan be cleaved by the 5′-exonuclease activity of Taq polymerase. Duringthe extension phase of the PCR amplification cycle, cleavage of theprobe by Taq polymerase releases the reporter dye from the remainder ofthe probe (and hence from the quencher moiety) and a sequence-specificfluorescent signal is generated. With each cycle, additional reporterdye molecules are cleaved from their respective probes, and thefluorescence intensity is monitored at regular intervals by laser opticsbuilt into the ABI PRISM™ Sequence Detection System. In each assay, aseries of parallel reactions containing serial dilutions of mRNA fromuntreated control samples generates a standard curve that is used toquantitate the percent inhibition after antisense oligonucleotidetreatment of test samples.

Prior to quantitative PCR analysis, primer-probe sets specific to thetarget gene being measured are evaluated for their ability to be“multiplexed” with a GAPDH amplification reaction. In multiplexing, boththe target gene and the internal standard gene GAPDH are amplifiedconcurrently in a single sample. In this analysis, mRNA isolated fromuntreated cells is serially diluted. Each dilution is amplified in thepresence of primer-probe sets specific for GAPDH only, target gene only(“single-plexing”), or both (multiplexing). Following PCR amplification,standard curves of GAPDH and target mRNA signal as a function ofdilution are generated from both the single-plexed and multiplexedsamples. If both the slope and correlation coefficient of the GAPDH andtarget signals generated from the multiplexed samples fall within 10% oftheir corresponding values generated from the single-plexed samples, theprimer-probe set specific for that target is deemed multiplexable. Othermethods of PCR are also known in the art.

PCR reagents were obtained from Invitrogen Corporation, (Carlsbad,Calif.). RT-PCR reactions were carried out by adding 20 μL PCR cocktail(2.5×PCR buffer minus MgCl₂, 6.6 mM MgCl₂, 375 μM each of dATP, dCTP,dCTP and dGTP, 375 nM each of forward primer and reverse primer, 125 nMof probe, 4 Units RNAse inhibitor, 1.25 Units PLATINUM® Taq, 5 UnitsMuLV reverse transcriptase, and 2.5×ROX dye) to 96-well platescontaining 30 μL total RNA solution (20-200 ng). The RT reaction wascarried out by incubation for 30 minutes at 48° C. Following a 10 minuteincubation at 95° C. to activate the PLATINUM® Taq, 40 cycles of atwo-step PCR protocol were carried out: 95° C. for 15 seconds(denaturation) followed by 60° C. for 1.5 minutes (annealing/extension).

Gene target quantities obtained by real time RT-PCR are normalized usingeither the expression level of GAPDH, a gene whose expression isconstant, or by quantifying total RNA using RiboGreen™ (MolecularProbes, Inc. Eugene, Oreg.). GAPDH expression is quantified by real timeRT-PCR, by being run simultaneously with the target, multiplexing, orseparately. Total RNA is quantified using RiboGreen™ RNA quantificationreagent (Molecular Probes, Inc. Eugene, Oreg.). Methods of RNAquantification by RiboGreen™ are taught in Jones, L. J., et al,(Analytical Biochemistry, 1998, 265, 368-374).

In this assay, 170 μL of RiboGreen™ working reagent (RiboGreen™ reagentdiluted 1:350 in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) is pipetted into a96-well plate containing 30 μL purified, cellular RNA. The plate is readin a CytoFluor 4000 (PE Applied Biosystems) with excitation at 485 nmand emission at 530 nm.

Probes and primers to human glucagon receptor were designed to hybridizeto a human glucagon receptor sequence, using published sequenceinformation (GenBank accession number NM_(—)000160.1, incorporatedherein as SEQ ID NO:4). For human glucagon receptor the PCR primerswere:

forward primer: GACACCCCCGCCAATACC (SEQ ID NO: 5)reverse primer: CCGCATCTCTTGAACACGAA (SEQ ID NO: 6) and the PCR probewas: FAM-TTGGCACCACAAAGT-TAMRA (SEQ ID NO: 7) where FAM is thefluorescent dye and TAMRA is the quencher dye. For human GAPDH the PCRprimers were:forward primer: GAAGGTGAAGGTCGGAGTC(SEQ ID NO:8)reverse primer: GAAGATGGTGATGGGATTTC (SEQ ID NO:9) and the PCR probewas: 5′ JOE-CAAGCTTCCCGTTCTCAGCC-TAMRA 3′ (SEQ ID NO: 10) where JOE isthe fluorescent reporter dye and TAMRA is the quencher dye.

Probes and primers to mouse glucagon receptor were designed to hybridizeto a mouse glucagon receptor sequence, using published sequenceinformation (GenBank accession number NM_(—)008101.1, incorporatedherein as SEQ ID NO: 11). For mouse glucagon receptor the PCR primerswere:

forward primer: ATTTCCTGCCCCTGGTACCT (SEQ ID NO:12)reverse primer: CGGGCCCACACCTCTTG (SEQ ID NO: 13) and the PCR probe was:FAM-CCACAAAGTGCAGCACCGCCTAGTGT-TAMRA (SEQ ID NO: 14) where FAM is thefluorescent reporter dye and TAMRA is the quencher dye. For mouse GAPDHthe PCR primers were:forward primer: GGCAAATTCAACGGCACAGT (SEQ ID NO:15)reverse primer: GGGTCTCGCTCCTGGAAGAT (SEQ ID NO:16) and the PCR probewas: 5′ JOE-AAGGCCGAGAATGGGAAGCTTGTCATC-TAMRA 3′ (SEQ ID NO: 17) whereJOE is the fluorescent reporter dye and TAMRA is the quencher dye.

Example 14 Northern Blot Analysis of Glucagon Receptor mRNA Levels

Eighteen hours after antisense treatment, cell monolayers were washedtwice with cold PBS and lysed in 1 mL RNAZOL™ (TEL-TEST “B” Inc.,Friendswood, Tex.). Total RNA was prepared following manufacturer'srecommended protocols. Twenty micrograms of total RNA was fractionatedby electrophoresis through 1.2% agarose gels containing 1.1%formaldehyde using a MOPS buffer system (AMRESCO, Inc. Solon, Ohio). RNAwas transferred from the gel to HYBOND™-N+ nylon membranes (AmershamPharmacia Biotech, Piscataway, N.J.) by overnight capillary transferusing a Northern/Southern Transfer buffer system (TEL-TEST “B” Inc.,Friendswood, Tex.). RNA transfer was confirmed by UV visualization.Membranes were fixed by UV cross-linking using a STRATALINKER™ UVCrosslinker 2400 (Stratagene, Inc, La Jolla, Calif.) and then probedusing QUICKHYB™ hybridization solution (Stratagene, La Jolla, Calif.)using manufacturer's recommendations for stringent conditions.

To detect human glucagon receptor, a human glucagon receptor specificprobe was prepared by PCR using the forward primer GACACCCCCGCCAATACC(SEQ ID NO: 5) and the reverse primer CCGCATCTCTTGAACACGAA (SEQ ID NO:6). To normalize for variations in loading and transfer efficiencymembranes were stripped and probed for human glyceraldehyde-3-phosphatedehydrogenase (GAPDH) RNA (Clontech, Palo Alto, Calif.).

To detect mouse glucagon receptor, a mouse glucagon receptor specificprobe was prepared by PCR using the forward primer ATTTCCTGCCCCTGGTACCT(SEQ ID NO: 12) and the reverse primer CGGGCCCACACCTCTTG (SEQ ID NO:13). To normalize for variations in loading and transfer efficiencymembranes were stripped and probed for mouse glyceraldehyde-3-phosphatedehydrogenase (GAPDH) RNA (Clontech, Palo Alto, Calif.).

Hybridized membranes were visualized and quantitated using aPHOSPHORIMAGER™ and IMAGEQUANT™ Software V3.3 (Molecular Dynamics,Sunnyvale, Calif.). Data was normalized to GAPDH levels in untreatedcontrols.

Example 15 Antisense Inhibition of Human Glucagon Receptor Expression byChimeric Phosphorothioate Oligonucleotides Having 2′-MOE Wings and aDeoxy Gap

In accordance with the present invention, a series of antisensecompounds were designed to target different regions of the humanglucagon receptor RNA, using published sequences (GenBank accessionnumber NM_(—)000160.1, incorporated herein as SEQ ID NO: 4, aconcatenation of three contigs from GenBank accession number AC069004.2,incorporated herein as SEQ ID NO: 18, and GenBank accession numberAJ245489.1, incorporated herein as SEQ ID NO: 19). The compounds areshown in Table 1. “Target site” indicates the first (5′-most) nucleotidenumber on the particular target sequence to which the compound binds.All compounds in Table 1 are chimeric oligonucleotides (“gapmers”) 20nucleotides in length, composed of a central “gap” region consisting often 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′directions) by five-nucleotide “wings”. The wings are composed of2′-methoxyethyl (2′-MOE)nucleotides. The internucleoside (backbone)linkages are phosphorothioate (P═S) throughout the oligonucleotide. Allcytidine residues are 5-methylcytidines. The compounds were analyzed fortheir effect on human glucagon receptor mRNA levels by quantitativereal-time PCR as described in other examples herein. Data are averagesfrom three experiments in which HepG2 cells were treated with theantisense oligonucleotides of the present invention. The positivecontrol for each datapoint is identified in the table by sequence IDnumber. If present, “N.D.” indicates “no data”.

TABLE 1 Inhibition of human glucagon receptor mRNA levels by chimericphosphorothioate oligonucleotides having 2′-MOE wings and a deoxy gapTARGET TARGET SEQ ISIS # REGION SEQ ID NO SITE SEQUENCE % INHIB ID NO310462 Coding 4 560 ccgcatctcttgaacacgaa 61  20 299881 5′UTR 4  97ttgagcctcagggcccgcgc 56  21 299882 5′UTR 4 121 gtgtcctcccctgaagctgc 68 22 299883 5′UTR 4 163 gagtggcagagcagcagagc 38  23 299884 5′UTR 4 192tgtgtgtgtacgctcctccg 76  24 299885 5′UTR 4 198 tcctggtgtgtgtgtacgct 67 25 299886 5′UTR 4 205 aatgcagtcctggtgtgtgt 30  26 299887 5′UTR 4 254ctgggcagctagctgcctcc 38  27 299888 Start Codon 4 263ggcatgcctctgggcagcta 73  28 299889 Coding 4 462 ccagcaggaatacttgtcga 43 29 299890 Coding 4 328 ggacctgtggctggcaggcc 72  30 299891 Coding 4 350aagtccatcacctgagcgga 39  31 299892 Coding 4 361 tctcaaacaggaagtccatc 37 32 299893 Coding 4 366 ccacttctcaaacaggaagt 24  33 299894 Coding 4 386cactggtcaccgtagagctt 31  34 299895 Coding 4 391 ggtgacactggtcaccgtag 40 35 299896 Coding 4 431 cacaccagctccgtgggagg 35  36 299897 Coding 4 442aggttctgttgcacaccagc 76  37 299898 Coding 4 453 atacttgtcgaaggttctgt 28 38 299899 Coding 4 539 cggtgttgcactttgtggtg 85  39 299900 5′UTR 19  546ccctggcagagacagcggca 79  40 299901 Coding 4 552 cttgaacacgaagcggtgtt 83 41 299902 Coding 4 564 gggcccgcatctcttgaaca 87  42 299903 intron: exon18  15279   cttctgcgagttacagtggc 58  43 junction 299904 Coding 4 651ctggacctcaatctcctcgc 43  44 299905 Coding 4 656 tccttctggacctcaatctc 5 45 299906 Coding 4 663 ggccacctccttctggacct 80  46 299907 Coding 4 669catcttggccacctccttct 39  47 299908 Coding 4 681 gaagctgctgtacatcttgg 71 48 299909 Coding 4 751 cccccaggatggccaaggcg 69  49 299910 Coding 4 830acggagctggctttcagcac 49  50 299911 Coding 4 866 ctgtagcgggtcctgagcag 48 51 299912 Coding 4 872 ttctggctgtagcgggtcct 54  52 299913 Coding 4 879gccaattttctggctgtagc 61  53 299914 Coding 4 889 tgaggtcgtcgccaattttc 56 54 299915 Coding 4 898 tgctgacactgaggtcgtcg 63  55 299916 Coding 4 904gccaggtgctgacactgagg 67  56 299917 Coding 4 966 cacgatgccatattgcatga 59 57 299918 Coding 4 1028  gtggccaggcccagcaggtt 52  58 299919 Coding 41122  cagacacttgaccactgccc 40  59 299920 Coding 4 1182 ccgcaggatccaccagaagc 46  60 299921 Coding 4 1210  tgatcaggatggccaggaag42  61 299922 Coding 4 1228  ggacgaagatgaagaagttg 44  62 299923 Coding 41259  cgcagcttggccacgagcag  8  63 299924 Coding 4 1274 tgcatctgccgtgcccgcag 58  64 299925 Coding 4 1291  acttgtagtctgtgtggtgc34  65 299926 Coding 4 1415  aggtcgaagaagagcttggc 38  66 299927 Coding 41528  gcactttgcccaggcgccag 78  67 299928 Coding 4 1539 ctcctcccatagcactttgc 40  68 299929 Coding 4 1608  aaactgcagctccttgctgg46  69 299930 Coding 4 1636  atgaatcctggctgccacca 70  70 299931 Coding 41670  ctagggaggccaccagccaa 49  71 299932 Coding 4 1681 tctcagccaatctagggagg 63  72 299933 Stop Codon 4 1704 tcccagcagggttcagaagg 30  73 299934 5′UTR 19  1747  ttcctgcaggtgacccaatg50  74 299935 3′UTR 4 1841  tctcgcagacagccacactg 43  75 299936 3′UTR 41854  agaggaggcccaatctcgca 79  76 299937 3′UTR 4 1881 tgcaccagggacaaggcagg  0  77 299938 3′UTR 4 1901  tggactcctctgctcacctc 58 78 299939 3′UTR 4 1938  tggcacgcagttcacggcac 54  79 299940 3′UTR 41969  acatgggacgtgccgacata 63  80 299941 3′UTR 4 1978 tttccatgcacatgggacgt 63  81 299942 3′UTR 4 1989  gttggaggacatttccatgc 79 82 299943 3′UTR 4 2015  cacggtgaccacttgagctc 18  83 299944 intron 18 11002   agatgtccgtgtttgtcagc  9  84 299945 intron 18  11557  taataactttttaaagaagg 17  85 299946 intron 18  12295  tactacgttgctcgggctgg 23  86 299947 intron 18  14121  agctctgtggctcagttacc 74  87 299948 intron: exon 18  15467  gtgcagcttgctgtggcaca 47  88 junction 299949 intron 18  16094cagcaaccgcttggtacagg 100   89 299950 intron: exon 18  17017agaagttgatctgtgtgaga 29  90 junction 299951 intron: exon 18  17456ccagcaggccctggagagac 53  91 junction 304471 5′UTR 4 100cctttgagcctcagggcccg 42  92 304472 5′UTR 4 103 gcccctttgagcctcagggc 25 93 304473 5′UTR 4 167 agctgagtggcagagcagca 76  94 304474 5′UTR 4 169gcagctgagtggcagagcag 75  95 304475 5′UTR 4 190 tgtgtgtacgctcctccgag 73 96 304476 5′UTR 4 194 ggtgtgtgtgtacgctcctc 72  97 304477 5′UTR 4 196ctggtgtgtgtgtacgctcc 71  98 304478 5′UTR 4 209 gggcaatgcagtcctggtgt 65 99 304479 5′UTR 4 246 ctagctgcctcccacatctg 54 100 304480 5′UTR 4 249cagctagctgcctcccacat 85 101 304481 5′UTR 4 257 cctctgggcagctagctgcc 44102 304482 Start Codon 4 262 gcatgcctctgggcagctag 62 103 304483 Coding 4325 cctgtggctggcaggccagc 68 104 304484 Coding 4 368 ttccacttctcaaacaggaa24 105 304485 Coding 4 370 gcttccacttctcaaacagg 49 106 304486 Coding 4375 gtagagcttccacttctcaa 41 107 304487 Coding 4 376 cgtagagcttccacttctca38 108 304488 Coding 4 395 ttgtggtgacactggtcacc 24 109 304489 Coding 4407 agcaggctcaggttgtggtg 52 110 304490 Coding 4 534 ttgcactttgtggtgccaag61 111 304491 Coding 4 535 gttgcactttgtggtgccaa 57 112 304492 Coding 4536 tgttgcactttgtggtgcca 67 113 304493 Coding 4 537 gtgttgcactttgtggtgcc75 114 304494 Coding 4 563 ggcccgcatctcttgaacac 87 115 304495 Coding 4567 gtcgggcccgcatctcttga 81 116 304496 Coding 4 617 tgggaggcatcacgccaagg60 117 304497 Coding 4 627 catctggcactgggaggcat 48 118 304498 Coding 4666 cttggccacctccttctgga 74 119 304499 Coding 4 671 tacatcttggccacctcctt24 120 304500 Coding 4 685 cctggaagctgctgtacatc 71 121 304501 Coding 4795 attcgcgtggatggcattgc 53 122 304502 Coding 4 848 agcccatcaatgaccagcac31 123 304503 Coding 4 861 gcgggtcctgagcagcccat 42 124 304504 Coding 4886 ggtcgtcgccaattttctgg 50 125 304505 Coding 4 893 acactgaggtcgtcgccaat22 126 304506 Coding 4 900 ggtgctgacactgaggtcgt 60 127 304507 Coding 4962 atgccatattgcatgaacac 27 128 304508 Coding 4 1032 gagggtggccaggcccagca 56 129 304509 Coding 4 1124  aacagacacttgaccactgc13 130 304510 Coding 4 1125  gaacagacacttgaccactg  8 131 304511 Coding 41158  gttgtcattgctggtccagc 65 132 304512 Coding 4 1168 agaagcccatgttgtcattg 44 133 304513 Coding 4 1187  gggaaccgcaggatccacca42 134 304514 Coding 4 1230  gcggacgaagatgaagaagt 54 135 304515 Coding 41638  agatgaatcctggctgccac 53 136 304516 3′UTR 4 1727 ccagagtccagccctagctg 41 137 304517 3′UTR 4 1732  gggtgccagagtccagccct 48138 304518 3′UTR 4 1735  tctgggtgccagagtccagc 65 139 304519 3′UTR 41736  ctctgggtgccagagtccag 75 140 304520 3′UTR 4 1737 cctctgggtgccagagtcca 74 141 304521 3′UTR 4 1740  acgcctctgggtgccagagt 55142 304522 3′UTR 4 1760  cagttctgggttgtccagcg 52 143 304523 3′UTR 41849  aggcccaatctcgcagacag 74 144 304524 3′UTR 4 1850 gaggcccaatctcgcagaca 80 145 304525 3′UTR 4 1856  ggagaggaggcccaatctcg 66146 304526 3′UTR 4 1861  tgcagggagaggaggcccaa 63 147 304527 3′UTR 41883  tctgcaccagggacaaggca 50 148 304528 3′UTR 4 1891 tgctcacctctgcaccaggg 66 149 304529 3′UTR 4 1893  tctgctcacctctgcaccag 32150 304530 3′UTR 4 1899  gactcctctgctcacctctg 31 151 304531 3′UTR 41905  gccctggactcctctgctca 69 152 304532 3′UTR 4 1932 gcagttcacggcacagcccc 53 153 304533 3′UTR 4 1933  cgcagttcacggcacagccc 30154 304534 3′UTR 4 1945  gggacactggcacgcagttc 61 155 304535 3′UTR 41971  gcacatgggacgtgccgaca 83 156 304536 3′UTR 4 1984 aggacatttccatgcacatg 61 157 304537 3′UTR 4 1986  ggaggacatttccatgcaca 69158 304538 3′UTR 4 1999  gctctttattgttggaggac 66 159 304539 3′UTR 42001  gagctctttattgttggagg 68 160 304540 3′UTR 4 2008 accacttgagctctttattg 40 161 304541 intron 18  3174  ggcagttttggcgtccccag67 162 304542 intron 18  6670  gagcttcctgcctcttcacg 39 163 304543 intron18  7544  ggataggatgtgcgtgtcta 42 164 304544 intron 18  7975 ctctctgcctccgatttctt 12 165 304545 intron: exon 18  14888  acaccagctctgcagggtag 75 166 junction 304546 intron: exon 18  15285  cacctccttctgcgagttac 33 167 junction 310441 Start Codon 4 258gcctctgggcagctagctgc 64 168 310442 Coding 4 317 tggcaggccagcagcagcag 87169 310443 Coding 4 321 tggctggcaggccagcagca 88 170 310444 Coding 4 347tccatcacctgagcggaggg 55 171 310445 Coding 4 351 gaagtccatcacctgagcgg 36172 310446 Coding 4 355 acaggaagtccatcacctga 28 173 310447 Coding 4 365cacttctcaaacaggaagtc 59 174 310448 Coding 4 389 tgacactggtcaccgtagag 18175 310449 Coding 4 393 gtggtgacactggtcaccgt 12 176 310450 Coding 4 397ggttgtggtgacactggtca 72 177 310451 Coding 4 403 ggctcaggttgtggtgacac 62178 310452 Coding 4 452 tacttgtcgaaggttctgtt 44 179 310453 Coding 4 458caggaatacttgtcgaaggt 40 180 310454 Coding 4 493 tgttggccgtggtattggcg 90181 310455 Coding 4 497 gagatgttggccgtggtatt 87 182 310456 Coding 4 500caggagatgttggccgtggt 95 183 310457 Coding 4 532 gcactttgtggtgccaaggc 96184 310458 Coding 4 540 gcggtgttgcactttgtggt 92 185 310459 Coding 4 544cgaagcggtgttgcactttg 50 186 310460 Coding 4 548 aacacgaagcggtgttgcac 87187 310461 Coding 4 556 atctcttgaacacgaagcgg 65 188 310463 Coding 4 588gggtccacgcacccactgac 50 189 310464 Coding 4 606 acgccaaggctgcccccggg 71190 310465 Coding 4 660 cacctccttctggacctcaa 31 191 310466 Coding 4 683tggaagctgctgtacatctt 57 192 310467 Coding 4 687 cacctggaagctgctgtaca 60193 310468 Coding 4 691 acatcacctggaagctgctg 73 194 310469 Coding 4 695gtgtacatcacctggaagct 79 195 310470 Coding 4 720 ccccagggacaggctgtagc 86196 310471 Coding 4 723 ggcccccagggacaggctgt 62 197 310472 Coding 4 860cgggtcctgagcagcccatc 48 198 310473 Coding 4 864 gtagcgggtcctgagcagcc 58199 310474 Coding 4 868 ggctgtagcgggtcctgagc 48 200 310475 Coding 4 919ccgctccatcactgagccag 52 201 310476 Coding 4 923 gccaccgctccatcactgag 41202 310477 Coding 4 951 catgaacaccgcggccacac 63 203 310478 Coding 4 955attgcatgaacaccgcggcc 76 204 310479 Coding 4 960 gccatattgcatgaacaccg 66205 310480 Coding 4 1019  cccagcaggttgtgcaggta 58 206 310481 Coding 41025  gccaggcccagcaggttgtg 72 207 310482 Coding 4 1029 ggtggccaggcccagcaggt 83 208 310483 Coding 4 1055  aggctgaagaagctcctctc71 209 310484 Coding 4 1059  gtagaggctgaagaagctcc 46 210 310485 Coding 41063  ccaggtagaggctgaagaag 25 211 310486 Coding 4 1068 gatgcccaggtagaggctga 51 212 310487 Coding 4 1072  agccgatgcccaggtagagg70 213 310488 Coding 4 1156  tgtcattgctggtccagcac 83 214 310489 Coding 41160  atgttgtcattgctggtcca 53 215 310490 Coding 4 1167 gaagcccatgttgtcattgc 45 216 310491 Coding 4 1173  ccaccagaagcccatgttgt50 217 310492 Coding 4 1176  gatccaccagaagcccatgt 53 218 310493 Coding 41185  gaaccgcaggatccaccaga 47 219 310494 Coding 4 1206 caggatggccaggaagacgg 39 220 310495 Coding 4 1209  gatcaggatggccaggaaga67 221 310496 Coding 4 1219  tgaagaagttgatcaggatg 10 222 310497 Coding 41222  agatgaagaagttgatcagg 20 223 310498 Coding 4 1287 gtagtctgtgtggtgcatct 35 224 310499 Coding 4 1290  cttgtagtctgtgtggtgca63 225 310500 Coding 4 1293  gaacttgtagtctgtgtggt 27 226 310501 Coding 41414  ggtcgaagaagagcttggcg 46 227 310502 Coding 4 1417 agaggtcgaagaagagcttg 26 228 310503 Coding 4 1423  tgaggaagaggtcgaagaag17 229 310504 Coding 4 1669  tagggaggccaccagccaag 53 230 315163 Coding 4686 acctggaagctgctgtacat 75 231 315164 Coding 4 409 gcagcaggctcaggttgtgg24 232 315165 Coding 4 1424  ctgaggaagaggtcgaagaa 42 233 315166 Coding 4398 aggttgtggtgacactggtc 34 234 315167 Coding 4 1212 gttgatcaggatggccagga 47 235 315168 Coding 4 1062  caggtagaggctgaagaagc40 236 315169 Coding 4 559 cgcatctcttgaacacgaag 48 237 315170 Coding 4543 gaagcggtgttgcactttgt 61 238 315171 Coding 4 454 aatacttgtcgaaggttctg16 239 315172 Coding 4 1026  ggccaggcccagcaggttgt 72 240 315173 Coding 41070  ccgatgcccaggtagaggct 59 241 315174 Coding 4 496agatgttggccgtggtattg 79 242 315175 Coding 4 399 caggttgtggtgacactggt 58243 315176 Coding 4 1420  ggaagaggtcgaagaagagc 26 244 315177 Coding 4392 tggtgacactggtcaccgta 49 245 315178 Coding 4 402 gctcaggttgtggtgacact62 246 315179 Coding 4 533 tgcactttgtggtgccaagg 75 247 315180 Coding 4689 atcacctggaagctgctgta 45 248 315181 Coding 4 956 tattgcatgaacaccgcggc78 249 315182 Coding 4 1208  atcaggatggccaggaagac 36 250 315183 Coding 4555 tctcttgaacacgaagcggt 71 251 315184 Coding 4 553 tcttgaacacgaagcggtgt87 252 315185 Coding 4 1027  tggccaggcccagcaggttg 61 253 315186 Coding 4871 tctggctgtagcgggtcctg 73 254 315187 Coding 4 498 ggagatgttggccgtggtat93 255 315188 Start Codon 4 259 tgcctctgggcagctagctg 70 256 315189Coding 4 1058  tagaggctgaagaagctcct 54 257 315190 Coding 4 348gtccatcacctgagcggagg 68 258 315191 Coding 4 1292  aacttgtagtctgtgtggtg39 259 315192 Stop Codon 4 1705  gtcccagcagggttcagaag 31 260 315193Coding 4 953 tgcatgaacaccgcggccac 73 261 315194 Coding 4 1024 ccaggcccagcaggttgtgc 73 262 315195 Coding 4 1061  aggtagaggctgaagaagct57 263 315196 Coding 4 1169  cagaagcccatgttgtcatt 47 264 315197 Coding 41161  catgttgtcattgctggtcc  0 265 315198 Coding 4 1021 ggcccagcaggttgtgcagg 84 266 315199 Coding 4 400 tcaggttgtggtgacactgg 42267 315200 Coding 4 1165  agcccatgttgtcattgctg 45 268 315201 Coding 4363 cttctcaaacaggaagtcca 47 269 315202 Coding 4 550 tgaacacgaagcggtgttgc83 270 315203 Coding 4 367 tccacttctcaaacaggaag 69 271 315204 Coding 4353 aggaagtccatcacctgagc 26 272 315205 Coding 4 1071 gccgatgcccaggtagaggc 82 273 315206 Coding 4 1186  ggaaccgcaggatccaccag36 274 315207 Coding 4 349 agtccatcacctgagcggag 63 275 315208 Coding 41221  gatgaagaagttgatcagga 28 276 315209 Coding 4 461cagcaggaatacttgtcgaa 27 277 315210 Coding 4 463 gccagcaggaatacttgtcg 41278 315211 Coding 4 320 ggctggcaggccagcagcag 72 279 315212 Coding 41183  accgcaggatccaccagaag 59 280 315213 Coding 4 862agcgggtcctgagcagccca 68 281 315214 Coding 4 565 cgggcccgcatctcttgaac 88282 315215 Coding 4 1295  cggaacttgtagtctgtgtg 29 283 315216 Coding 41177  ggatccaccagaagcccatg 58 284 315217 Stop Codon 4 1706 ggtcccagcagggttcagaa 34 285 315218 Coding 4 1184  aaccgcaggatccaccagaa55 286 315219 Coding 4 410 ggcagcaggctcaggttgtg 50 287 315220 Coding 4495 gatgttggccgtggtattgg 86 288 315221 Coding 4 455 gaatacttgtcgaaggttct37 289 315222 Coding 4 1215  gaagttgatcaggatggcca 39 290 315223 Coding 4688 tcacctggaagctgctgtac 48 291 315224 Coding 4 959 ccatattgcatgaacaccgc20 292 315225 Coding 4 863 tagcgggtcctgagcagccc 61 293 315226 5′UTR 4256 ctctgggcagctagctgcct 28 294 315227 Coding 4 359 tcaaacaggaagtccatcac17 295 315228 Coding 4 1172  caccagaagcccatgttgtc 15 296 315229 Coding 4694 tgtacatcacctggaagctg 67 297 315230 Coding 4 494 atgttggccgtggtattggc52 298 315231 Coding 4 1069  cgatgcccaggtagaggctg  7 299 315232 Coding 41178  aggatccaccagaagcccat 83 300 315233 Coding 4 1207 tcaggatggccaggaagacg 52 301 315234 Coding 4 352 ggaagtccatcacctgagcg 60302 315235 Start Codon 4 261 catgcctctgggcagctagc 65 303 315236 Coding 4561 cccgcatctcttgaacacga 51 304 315237 Coding 4 323 tgtggctggcaggccagcag60 305 315238 Coding 4 324 ctgtggctggcaggccagca 43 306 315239 Coding 41179  caggatccaccagaagccca 88 307 315240 Coding 4 1223 aagatgaagaagttgatcag  0 308 315241 Coding 4 1289  ttgtagtctgtgtggtgcat66 309 315242 Coding 4 322 gtggctggcaggccagcagc 47 310 315243 Coding 4406 gcaggctcaggttgtggtga 44 311 315244 Coding 4 870 ctggctgtagcgggtcctga61 312 315245 5′UTR 4 255 tctgggcagctagctgcctc 24 313 315246 Coding 4464 ggccagcaggaatacttgtc 71 314 315247 Coding 4 360 ctcaaacaggaagtccatca13 315 315248 Coding 4 1060  ggtagaggctgaagaagctc 49 316 315249 Coding 41422  gaggaagaggtcgaagaaga 69 317 315250 Coding 4 1416 gaggtcgaagaagagcttgg 32 318 315251 Coding 4 1288  tgtagtctgtgtggtgcatc30 319 315252 Coding 4 1216  agaagttgatcaggatggcc 17 320 315253 Coding 4542 aagcggtgttgcactttgtg 55 321 315254 Coding 4 456 ggaatacttgtcgaaggttc44 322 315255 Coding 4 1419  gaagaggtcgaagaagagct 34 323 315256 Coding 4460 agcaggaatacttgtcgaag 10 324 315257 Coding 4 404 aggctcaggttgtggtgaca58 325 315258 Coding 4 538 ggtgttgcactttgtggtgc 58 326 315259 Coding 41294  ggaacttgtagtctgtgtgg 30 327 315260 Coding 4 390gtgacactggtcaccgtaga 19 328 315261 Coding 4 954 ttgcatgaacaccgcggcca 59329 315262 Coding 4 684 ctggaagctgctgtacatct 61 330 315263 Coding 41174  tccaccagaagcccatgttg  1 331 315264 Coding 4 1214 aagttgatcaggatggccag 44 332 315265 Coding 4 1023  caggcccagcaggttgtgca51 333 315266 Coding 4 920 accgctccatcactgagcca 38 334 315267 Coding 41220  atgaagaagttgatcaggat  0 335 315268 Coding 4 554ctcttgaacacgaagcggtg 78 336 315269 Coding 4 318 ctggcaggccagcagcagca 37337 315270 Coding 4 499 aggagatgttggccgtggta 97 338 315271 Coding 41164  gcccatgttgtcattgctgg 66 339 315272 Coding 4 1217 aagaagttgatcaggatggc 25 340 315273 Coding 4 1064  cccaggtagaggctgaagaa62 341 315274 Coding 4 1163  cccatgttgtcattgctggt 55 342 315275 Coding 4547 acacgaagcggtgttgcact 46 343 315276 Coding 4 408 cagcaggctcaggttgtggt62 344 315277 Coding 4 394 tgtggtgacactggtcaccg 15 345 315278 Coding 41020  gcccagcaggttgtgcaggt 83 346 315279 Coding 4 869tggctgtagcgggtcctgag 33 347 315280 Coding 4 562 gcccgcatctcttgaacacg 77348 315281 Coding 4 1418  aagaggtcgaagaagagctt 40 349 315282 Coding 4411 gggcagcaggctcaggttgt 23 350 315283 Coding 4 557 catctcttgaacacgaagcg40 351 315284 Coding 4 1175  atccaccagaagcccatgtt 38 352 315285 Coding 41155  gtcattgctggtccagcact 75 353 315286 Coding 4 566tcgggcccgcatctcttgaa 74 354 315287 Coding 4 721 cccccagggacaggctgtag 53355 315288 Coding 4 1162  ccatgttgtcattgctggtc 43 356 315289 Coding 41056  gaggctgaagaagctcctct  2 357 315290 Coding 4 549gaacacgaagcggtgttgca 88 358 315291 Coding 4 362 ttctcaaacaggaagtccat  9359 315292 Coding 4 1159  tgttgtcattgctggtccag 47 360 315293 Coding 4457 aggaatacttgtcgaaggtt 55 361 315294 Coding 4 405 caggctcaggttgtggtgac38 362 315295 Coding 4 1421  aggaagaggtcgaagaagag 19 363 315296 Coding 41425  gctgaggaagaggtcgaaga 33 364 315297 Coding 4 546cacgaagcggtgttgcactt 81 365 315298 Coding 4 1166  aagcccatgttgtcattgct35 366 315299 Start Codon 4 260 atgcctctgggcagctagct 63 367 315300Coding 4 690 catcacctggaagctgctgt 63 368 315301 Coding 4 364acttctcaaacaggaagtcc 31 369 315302 Coding 4 558 gcatctcttgaacacgaagc 44370 315303 Coding 4 958 catattgcatgaacaccgcg 48 371 315304 Coding 41170  ccagaagcccatgttgtcat 33 372 315305 Coding 4 867gctgtagcgggtcctgagca 50 373 315306 Coding 4 865 tgtagcgggtcctgagcagc 62374 315307 Coding 4 1022  aggcccagcaggttgtgcag 37 375 315308 Coding 4692 tacatcacctggaagctgct 41 376 315309 Coding 4 1181 cgcaggatccaccagaagcc 49 377 315310 Coding 4 357 aaacaggaagtccatcacct 21378 315311 Coding 4 1057  agaggctgaagaagctcctc 49 379 315312 Coding 41211  ttgatcaggatggccaggaa 54 380 315313 Coding 4 541agcggtgttgcactttgtgg 81 381 315314 Coding 4 319 gctggcaggccagcagcagc 75382 315315 Coding 4 545 acgaagcggtgttgcacttt 68 383 315316 Coding 4 952gcatgaacaccgcggccaca 80 384 315317 Coding 4 354 caggaagtccatcacctgag 26385 315318 Coding 4 1180  gcaggatccaccagaagccc 72 386 315319 Coding 41213  agttgatcaggatggccagg 33 387 315320 Coding 4 722gcccccagggacaggctgta 51 388 315321 Coding 4 356 aacaggaagtccatcacctg  0389 315322 Coding 4 957 atattgcatgaacaccgcgg 56 390 315323 Coding 4 459gcaggaatacttgtcgaagg 59 391 315324 Coding 4 693 gtacatcacctggaagctgc 79392 315325 Coding 4 1153  cattgctggtccagcactgg 61 393 315326 Coding 4358 caaacaggaagtccatcacc 10 394 315327 Coding 4 1031 agggtggccaggcccagcag 27 395 315328 Coding 4 551 ttgaacacgaagcggtgttg 66396 315329 Coding 4 1171  accagaagcccatgttgtca 47 397 315330 Coding 4401 ctcaggttgtggtgacactg 14 398 315331 Coding 4 396 gttgtggtgacactggtcac 5 399

As shown in Table 1, SEQ ID NOs 20, 21, 22, 24, 25, 28, 29, 30, 35, 37,39, 40, 41, 42, 43, 44, 46, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 64, 67, 68, 69, 70, 71, 72, 74, 75, 76, 78, 79, 80, 81,82, 87, 88, 89, 91, 92, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104,106, 107, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 121, 122,124, 125, 127, 129, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141,142, 143, 144, 145, 146, 147, 148, 149, 152, 153, 155, 156, 157, 158,159, 160, 161, 162, 164, 166, 168, 169, 170, 171, 174, 177, 178, 179,180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 192, 193, 194,195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208,209, 210, 212, 213, 214, 215, 216, 217, 218, 219, 221, 225, 227, 230,231, 233, 235, 236, 237, 238, 240, 241, 242, 243, 245, 246, 247, 248,249, 251, 252, 253, 254, 255, 256, 257, 258, 261, 262, 263, 264, 266,267, 268, 269, 270, 271, 273, 275, 278, 279, 280, 281, 282, 284, 286,287, 288, 291, 293, 297, 298, 300, 301, 302, 303, 304, 305, 306, 307,309, 310, 311, 312, 314, 316, 317, 321, 322, 325, 326, 329, 330, 332,333, 336, 338, 339, 341, 342, 343, 344, 346, 348, 349, 351, 353, 354,355, 356, 358, 360, 361, 365, 367, 368, 370, 371, 373, 374, 376, 377,379, 380, 381, 382, 383, 384, 386, 388, 390, 391, 392, 393, 396 and 397demonstrated at least 40% inhibition of human glucagon receptorexpression in this assay and are therefore preferred. SEQ ID NO: 183,184, 231, 249, 254, 346, 365 and 392 are presently more preferred. Thetarget regions to which the preferred sequences are complementary areherein referred to as “preferred target segments” and are thereforepreferred for targeting by compounds of the present invention. Thesepreferred target segments are shown in Table 3. These sequences areshown to contain thymine (T) but one of skill in the art will appreciatethat thymine (T) is generally replaced by uracil (U) in RNA sequences.The sequences represent the reverse complement of the preferredantisense compounds shown in Table 1. “Target site” indicates the first(5′-most) nucleotide number on the particular target nucleic acid towhich the oligonucleotide binds. Also shown in Table 3 is the species inwhich each of the preferred target segments was found.

Example 16 Antisense Inhibition of Mouse Glucagon Receptor Expression byChimeric Phosphorothioate Oligonucleotides Having 2′-MOE Wings and aDeoxy Gap

In accordance with the present invention, a second series of antisensecompounds were designed to target different regions of the mouseglucagon receptor RNA, using published sequences (GenBank accessionnumber NM_(—)008101.1, incorporated herein as SEQ ID NO: 11, an mRNAsequence derived from GenBank accession number AF229079.1 with analternate promoter, incorporated herein as SEQ ID NO: 400, GenBankaccession number AF229079.1, incorporated herein as SEQ ID NO: 401, asecond mRNA sequence derived from GenBank accession number AF229079.1with an alternate promoter, incorporated herein as SEQ ID NO: 402, andGenBank accession number AA920726.1, incorporated herein as SEQ ID NO:403). The compounds are shown in Table 2. “Target site” indicates thefirst (5′-most) nucleotide number on the particular target nucleic acidto which the compound binds. All compounds in Table 2 are chimericoligonucleotides (“gapmers”) 20 nucleotides in length, composed of acentral “gap” region consisting of ten 2′-deoxynucleotides, which isflanked on both sides (5′ and 3′ directions) by five-nucleotide “wings”.The wings are composed of 2′-methoxyethyl (2′-MOE)nucleotides. Theinternucleoside (backbone) linkages are phosphorothioate (P═S)throughout the oligonucleotide. All cytidine residues are5-methylcytidines. The compounds were analyzed for their effect on mouseglucagon receptor mRNA levels by quantitative real-time PCR as describedin other examples herein. Data are averages from three experiments inwhich mouse primary hepatocytes were treated with the antisenseoligonucleotides of the present invention. The positive control for eachdatapoint is identified in the table by sequence ID number. If present,“N.D.” indicates “no data”.

TABLE 2 Inhibition of mouse glucagon receptor mRNA levels by chimericphosphorothioate oligonucleotides having 2′-MOE wings and a deoxy gapTARGET TARGET CONTROL ISIS # REGION SEQ ID NO SITE SEQUENCE % INHIB SEQID NO SEQ ID NO 148350 5′UTR 11   57 cccacatctggcagaggttg 30 404 1148355 coding 11  182 ttctcaaacaaaaagtccat  7 405 1 148356 coding 11 193 agagcttccacttctcaaac 63 406 1 148357 coding 11  203tggtcactatagagcttcca 39 407 1 148359 coding 11  227 agcaggcttaggttgtggtg39 408 1 148363 coding 11  322 ggcaggaaatgttggcagtg 52 409 1 148366coding 11  383 ggcccacacctcttgaacac 93 410 1 148368 exon: exon 11  477ccccttctggacctcgatct 45 411 1 junction 148371 coding 11  538ccagggacagactgtagccc 53 412 1 148372 exon: exon 11  589agtgcagcttcctgaggccc 52 413 1 junction 148381 coding 11  938cacttgaccaccacccaggg 47 414 1 148382 coding 11  947 tcaaacagacacttgaccac 0 415 1 148385 coding 11  977 ttgtcattgctggtccagca 57 416 1 148387coding 11  998 aggatccaccagaatcccat 53 417 1 148390 coding 11 1139agggtcagcgtggacctggc 45 418 1 148393 coding 11 1226 aagagcttggtggagcgcag26 419 1 148394 coding 11 1277 tagagaacagccaccagcag 26 420 1 148395coding 11 1285 ggaaacagtagagaacagcc 26 421 1 148396 exon: exon 11 1299cacctccttgttgaggaaac  0 422 1 junction 180446 5′UTR 11    7ctcctcaggttgcaagggag 15 423 1 180447 5′UTR 11   14 tgcacctctcctcaggttgc38 424 1 180448 5′UTR 11   25 ctcagagtgtgtgcacctct 54 425 1 180449 5′UTR11   30 aggtcctcagagtgtgtgca 55 426 1 180450 5′UTR 11   48ggcagaggttgcacacctag 39 427 1 180451 Start Codon 11   80ggcatgcctctgggtagcca 40 428 1 180452 Coding 11  141 tggcagacatgacagcacca 5 429 1 180453 Coding 11  192 gagcttccacttctcaaaca 37 430 1 180454Coding 11  251 cagaccagctcagtaggtgg 45 431 1 180455 Coding 11  291ggtgtcaggccagcaggagt 58 432 1 180456 Coding 11  359 cggtgctgcactttgtggca68 433 1 180457 Coding 11  371 ttgaacactaggcggtgctg 69 434 1 180458Coding 11  410 cgtggccctcgaacccactg 39 435 1 180459 Coding 11  545aaggcccccagggacagact 56 436 1 180460 Coding 11  572 cccagcaggatgaccagcgc59 437 1 180461 Coding 11  582 cttcctgaggcccagcagga 40 438 1 180462Coding 11  650 acagagccagccttgagcac 47 439 1 180463 Coding 11  764actgtggccactctgcagcc 43 440 1 180464 Coding 11  775 actgcatgatcactgtggcc60 441 1 180465 Coding 11  785 atgatgccgtactgcatgat 58 442 1 180466Coding 11  836 agcaggctgtacaggtacac 36 443 1 180467 Coding 11  974tcattgctggtccagcactg 62 444 1 180468 Coding 11 1011 gacaggaatacgcaggatcc 0 445 1 180469 Coding 11 1079 cgcagcttggccacaagaag 56 446 1 180470Coding 11 1090 tctgatgggcacgcagcttg  8 447 1 180471 Coding 11 1100gcatagtgcatctgatgggc 45 448 1 180472 Coding 11 1110 cttgtaatcagcatagtgca14 449 1 180473 Coding 11 1256 ccctggaaggagctgaggaa 45 450 1 180474Coding 11 1292 ttgttgaggaaacagtagag 47 451 1 180475 Coding 11 1348gagctttgccttcttgccat 64 452 1 180476 Coding 11 1360 tttcctcctgaagagctttg56 453 1 180477 Coding 11 1388 atgtggctgccatggctgct 64 454 1 180478Coding 11 1435 gctgaagtttctcacaggga 56 455 1 180479 Coding 11 1450tgcctgcactcataagctga 48 456 1 180480 Coding 11 1470 acagccagtcccactgctgc41 457 1 180481 Coding 11 1512 ccttgggagactactggcca 56 458 1 180482 StopCodon 11 1544 caagtggagattcaggtggg 47 459 1 180483 3′UTR 11 1567ttgaacacaacctgcctagg  9 460 1 180484 3′UTR 11 1575 gccctttcttgaacacaacc34 461 1 180485 3′UTR 11 1600 atctggctctgggttgtcct 59 462 1 180486 3′UTR11 1610 ttggccgggcatctggctct 53 463 1 180487 3′UTR 11 1620ctcttcaaccttggccgggc 66 464 1 180488 3′UTR 11 1646 tacaagctgctgtcttgctg54 465 1 180489 3′UTR 11 1687 ggcctgtgccaggctaggac 47 466 1 180490 3′UTR11 1724 gcttctccatcatatccaac 47 467 1 180491 3′UTR 11 1750aacactcagagttcatagat 51 468 1 180492 3′UTR 11 1756 catgggaacactcagagttc49 469 1 180493 3′UTR 11 1795 ctgaaggacatatctgggta 51 470 1 180494intron 401  3953 gtaacaaaggcgagaccaag 36 471 1 180495 intron 401  5396gaggaagtgtcaccattagg 23 472 1 180496 intron: exon 401  7321cagaccagctctgtgaaggt 32 473 1 junction 180497 intron: exon 401  7505cggtgctgcactgggcatgg 77 474 1 junction 180498 exon: intron 401  8075ctgggctcaccccgtcactg 27 475 1 junction 180499 intron 401  8766ccaaggatgggcaacctgac 33 476 1 180500 exon: intron 401  9005ccttaccaaccggaacttgt  2 477 1 junction 180501 genomic 402   128cctctcctcaggtgtgctca  3 478 1 180502 genomic 400    10ccaagcccaaggcctcatga 40 479 1 180503 genomic 400    85ctcaggctgcagaggaccag 39 480 1 180504 genomic 403    40taggtctcttccctccactc  4 481 1

As shown in Table 2, SEQ ID NOs 404, 406, 407, 408, 409, 410, 411, 412,413, 414, 416, 417, 418, 424, 425, 426, 427, 428, 430, 431, 432, 433,434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 446, 448, 450,451, 452, 453, 454, 455, 456, 457, 458, 459, 461, 462, 463, 464, 465,466, 467, 468, 469, 470, 471, 473, 474, 476, 479 and 480 demonstrated atleast 30% inhibition of mouse glucagon receptor expression in thisexperiment and are therefore preferred. The target regions to whichthese preferred sequences are complementary are herein referred to as“preferred target segments” and are therefore preferred for targeting bycompounds of the present invention. These preferred target segments areshown in Table 3. These sequences are shown to contain thymine (T) butone of skill in the art will appreciate that thymine (T) is generallyreplaced by uracil (U) in RNA sequences. The sequences represent thereverse complement of the preferred antisense compounds shown in Tables1 and 2. “Target site” indicates the first (5′-most) nucleotide numberon the particular target nucleic acid to which the oligonucleotidebinds. Also shown in Table 3 is the species in which each of thepreferred target segments was found.

TABLE 3 Sequence and position of preferred target segments identified inglucagon receptor. TARGET TARGET REV COMP SITE ID SEQ ID NO SITESEQUENCE OF SEQ ID ACTIVE IN SEQ ID NO 215734 4  97 gcgcgggccctgaggctcaa 21 H. sapiens 484 215735 4 121 gcagcttcaggggaggacac  22 H. sapiens 485215737 4 192 cggaggagcgtacacacaca  24 H. sapiens 486 215738 4 198agcgtacacacacaccagga  25 H. sapiens 487 110316 4 263tagctgcccagaggcatgcc  28 H. sapiens 488 215800 4 462tcgacaagtattcctgctgg  29 H. sapiens 489 215741 4 328ggcctgccagccacaggtcc  30 H. sapiens 490 215746 4 391ctacggtgaccagtgtcacc  35 H. sapiens 491 215748 4 442gctggtgtgcaacagaacct  37 H. sapiens 492 110318 4 539caccacaaagtgcaacaccg  39 H. sapiens 493 215798 19  546tgccgctgtctctgccaggg  40 H. sapiens 494 215750 4 552aacaccgcttcgtgttcaag  41 H. sapiens 495 215751 4 564tgttcaagagatgcgggccc  42 H. sapiens 496 215801 18  15279  gccactgtaactcgcagaag  43 H. sapiens 497 215752 4 651gcgaggagattgaggtccag  44 H. sapiens 498 215754 4 663aggtccagaaggaggtggcc  46 H. sapiens 499 215756 4 681ccaagatgtacagcagcttc  48 H. sapiens 500 215757 4 751cgccttggccatcctggggg  49 H. sapiens 501 215758 4 830gtgctgaaagccagctccgt  50 H. sapiens 502 215759 4 866ctgctcaggacccgctacag  51 H. sapiens 503 215760 4 872aggacccgctacagccagaa  52 H. sapiens 504 215761 4 879gctacagccagaaaattggc  53 H. sapiens 505 215762 4 889gaaaattggcgacgacctca  54 H. sapiens 506 215763 4 898cgacgacctcagtgtcagca  55 H. sapiens 507 215764 4 904cctcagtgtcagcacctggc  56 H. sapiens 508 215765 4 966tcatgcaatatggcatcgtg  57 H. sapiens 509 215766 4 1028 aacctgctgggcctggccac  58 H. sapiens 510 215767 4 1122 gggcagtggtcaagtgtctg  59 H. sapiens 511 215768 4 1182 gcttctggtggatcctgcgg  60 H. sapiens 512 215769 4 1210 cttcctggccatcctgatca  61 H. sapiens 513 215770 4 1228 caacttcttcatcttcgtcc  62 H. sapiens 514 215771 4 1274 ctgcgggcacggcagatgca  64 H. sapiens 515 215774 4 1528 ctggcgcctgggcaaagtgc  67 H. sapiens 516 215775 4 1539 gcaaagtgctatgggaggag  68 H. sapiens 517 215776 4 1608 ccagcaaggagctgcagttt  69 H. sapiens 518 215777 4 1636 tggtggcagccaggattcat  70 H. sapiens 519 215778 4 1670 ttggctggtggcctccctag  71 H. sapiens 520 215779 4 1681 cctccctagattggctgaga  72 H. sapiens 521 215799 19  1747 cattgggtcacctgcaggaa  74 H. sapiens 522 215781 4 1841 cagtgtggctgtctgcgaga  75 H. sapiens 523 215782 4 1854 tgcgagattgggcctcctct  76 H. sapiens 524 215784 4 1901 gaggtgagcagaggagtcca  78 H. sapiens 525 215785 4 1938 gtgccgtgaactgcgtgcca  79 H. sapiens 526 215786 4 1969 tatgtcggcacgtcccatgt  80 H. sapiens 527 215787 4 1978 acgtcccatgtgcatggaaa  81 H. sapiens 528 215788 4 1989 gcatggaaatgtcctccaac  82 H. sapiens 529 215793 18  14121  ggtaactgagccacagagct  87 H. sapiens 530 215794 18  15467  tgtgccacagcaagctgcac  88 H. sapiens 531 215795 18  16094  cctgtaccaagcggttgctg  89 H. sapiens 532 215797 18  17456  gtctctccagggcctgctgg  91 H. sapiens 533 220245 4 100cgggccctgaggctcaaagg  92 H. sapiens 534 220247 4 167tgctgctctgccactcagct  94 H. sapiens 535 220248 4 169ctgctctgccactcagctgc  95 H. sapiens 536 220249 4 190ctcggaggagcgtacacaca  96 H. sapiens 537 220250 4 194gaggagcgtacacacacacc  97 H. sapiens 538 220251 4 196ggagcgtacacacacaccag  98 H. sapiens 539 220252 4 209acaccaggactgcattgccc  99 H. sapiens 540 220253 4 246cagatgtgggaggcagctag 100 H. sapiens 541 220254 4 249atgtgggaggcagctagctg 101 H. sapiens 542 220255 4 257ggcagctagctgcccagagg 102 H. sapiens 543 220256 4 262ctagctgcccagaggcatgc 103 H. sapiens 544 220257 4 325gctggcctgccagccacagg 104 H. sapiens 545 220259 4 370cctgtttgagaagtggaagc 106 H. sapiens 546 220260 4 375ttgagaagtggaagctctac 107 H. sapiens 547 110282 4 407caccacaacctgagcctgct 110 H. sapiens 548 220263 4 534cttggcaccacaaagtgcaa 111 H. sapiens 549 220264 4 535ttggcaccacaaagtgcaac 112 H. sapiens 550 220265 4 536 tggcaccacaaagtgcaaca113 H. sapiens 551 220266 4 537 ggcaccacaaagtgcaacac 114 H. sapiens 552110289 4 563 gtgttcaagagatgcgggcc 115 H. sapiens 553 220267 4 567tcaagagatgcgggcccgac 116 H. sapiens 554 220268 4 617ccttggcgtgatgcctccca 117 H. sapiens 555 220269 4 627atgcctcccagtgccagatg 118 H. sapiens 556 220270 4 666tccagaaggaggtggccaag 119 H. sapiens 557 220272 4 685gatgtacagcagcttccagg 121 H. sapiens 558 220273 4 795gcaatgccatccacgcgaat 122 H. sapiens 559 220275 4 861atgggctgctcaggacccgc 124 H. sapiens 560 220276 4 886ccagaaaattggcgacgacc 125 H. sapiens 561 220277 4 900acgacctcagtgtcagcacc 127 H. sapiens 562 220279 4 1032 tgctgggcctggccaccctc 129 H. sapiens 563 220282 4 1158 gctggaccagcaatgacaac 132 H. sapiens 564 220283 4 1168 caatgacaacatgggcttct 133 H. sapiens 565 220284 4 1187 tggtggatcctgcggttccc 134 H. sapiens 566 220285 4 1230 acttcttcatcttcgtccgc 135 H. sapiens 567 220286 4 1638 gtggcagccaggattcatct 136 H. sapiens 568 220287 4 1727 cagctagggctggactctgg 137 H. sapiens 569 220288 4 1732 agggctggactctggcaccc 138 H. sapiens 570 220289 4 1735 gctggactctggcacccaga 139 H. sapiens 571 220290 4 1736 ctggactctggcacccagag 140 H. sapiens 572 220291 4 1737 tggactctggcacccagagg 141 H. sapiens 573 220292 4 1740 actctggcacccagaggcgt 142 H. sapiens 574 220293 4 1760 cgctggacaacccagaactg 143 H. sapiens 575 220294 4 1849 ctgtctgcgagattgggcct 144 H. sapiens 576 220295 4 1850 tgtctgcgagattgggcctc 145 H. sapiens 577 220296 4 1856 cgagattgggcctcctctcc 146 H. sapiens 578 220297 4 1861 ttgggcctcctctccctgca 147 H. sapiens 579 220298 4 1883 tgccttgtccctggtgcaga 148 H. sapiens 580 220299 4 1891 ccctggtgcagaggtgagca 149 H. sapiens 581 220302 4 1905 tgagcagaggagtccagggc 152 H. sapiens 582 220303 4 1932 ggggctgtgccgtgaactgc 153 H. sapiens 583 220305 4 1945 gaactgcgtgccagtgtccc 155 H. sapiens 584 220306 4 1971 tgtcggcacgtcccatgtgc 156 H. sapiens 585 220307 4 1984 catgtgcatggaaatgtcct 157 H. sapiens 586 220308 4 1986 tgtgcatggaaatgtcctcc 158 H. sapiens 587 220309 4 1999 gtcctccaacaataaagagc 159 H. sapiens 588 220310 4 2001 cctccaacaataaagagctc 160 H. sapiens 589 220311 4 2008 caataaagagctcaagtggt 161 H. sapiens 590 220312 18  3174 ctggggacgccaaaactgcc 162 H. sapiens 591 220314 18  7544 tagacacgcacatcctatcc 164 H. sapiens 592 220316 18  14888  ctaccctgcagagctggtgt 166 H. sapiens 593 226083 4 258gcagctagctgcccagaggc 168 H. sapiens 594 226084 4 317ctgctgctgctggcctgcca 169 H. sapiens 595 226085 4 321tgctgctggcctgccagcca 170 H. sapiens 596 226086 4 347ccctccgctcaggtgatgga 171 H. sapiens 597 226089 4 365gacttcctgtttgagaagtg 174 H. sapiens 598 110281 4 397tgaccagtgtcaccacaacc 177 H. sapiens 599 226092 4 403gtgtcaccacaacctgagcc 178 H. sapiens 600 226093 4 452aacagaaccttcgacaagta 179 H. sapiens 601 226094 4 458accttcgacaagtattcctg 180 H. sapiens 602 226095 4 493cgccaataccacggccaaca 181 H. sapiens 603 226096 4 497aataccacggccaacatctc 182 H. sapiens 604 226097 4 500accacggccaacatctcctg 183 H. sapiens 605 226098 4 532gccttggcaccacaaagtgc 184 H. sapiens 606 110288 4 540accacaaagtgcaacaccgc 185 H. sapiens 607 226099 4 544caaagtgcaacaccgcttcg 186 H. sapiens 608 226100 4 548gtgcaacaccgcttcgtgtt 187 H. sapiens 609 226101 4 556ccgcttcgtgttcaagagat 188 H. sapiens 610 226103 4 588gtcagtgggtgcgtggaccc 189 H. sapiens 611 226104 4 606cccgggggcagccttggcgt 190 H. sapiens 612 226106 4 683aagatgtacagcagcttcca 192 H. sapiens 613 226107 4 687tgtacagcagcttccaggtg 193 H. sapiens 614 226108 4 691cagcagcttccaggtgatgt 194 H. sapiens 615 226109 4 695agcttccaggtgatgtacac 195 H. sapiens 616 226110 4 720gctacagcctgtccctgggg 196 H. sapiens 617 226111 4 723acagcctgtccctgggggcc 197 H. sapiens 618 226112 4 860gatgggctgctcaggacccg 198 H. sapiens 619 226113 4 864ggctgctcaggacccgctac 199 H. sapiens 620 226114 4 868gctcaggacccgctacagcc 200 H. sapiens 621 226115 4 919ctggctcagtgatggagcgg 201 H. sapiens 622 226116 4 923ctcagtgatggagcggtggc 202 H. sapiens 623 226117 4 951gtgtggccgcggtgttcatg 203 H. sapiens 624 226118 4 955ggccgcggtgttcatgcaat 204 H. sapiens 625 226119 4 960cggtgttcatgcaatatggc 205 H. sapiens 626 226120 4 1019 tacctgcacaacctgctggg 206 H. sapiens 627 226121 4 1025 cacaacctgctgggcctggc 207 H. sapiens 628 226122 4 1029 acctgctgggcctggccacc 208 H. sapiens 629 226123 4 1055 gagaggagcttcttcagcct 209 H. sapiens 630 226124 4 1059 ggagcttcttcagcctctac 210 H. sapiens 631 226126 4 1068 tcagcctctacctgggcatc 212 H. sapiens 632 110302 4 1072 cctctacctgggcatcggct 213 H. sapiens 633 226127 4 1156 gtgctggaccagcaatgaca 214 H. sapiens 634 226128 4 1160 tggaccagcaatgacaacat 215 H. sapiens 635 226129 4 1167 gcaatgacaacatgggcttc 216 H. sapiens 636 226130 4 1173 acaacatgggcttctggtgg 217 H. sapiens 637 226131 4 1176 acatgggcttctggtggatc 218 H. sapiens 638 226132 4 1185 tctggtggatcctgcggttc 219 H. sapiens 639 226134 4 1209 tcttcctggccatcctgatc 221 H. sapiens 640 226138 4 1290 tgcaccacacagactacaag 225 H. sapiens 641 226140 4 1414 cgccaagctcttcttcgacc 227 H. sapiens 642 226143 4 1669 cttggctggtggcctcccta 230 H. sapiens 643 231032 4 686atgtacagcagcttccaggt 231 H. sapiens 644 231034 4 1424 ttcttcgacctcttcctcag 233 H. sapiens 645 231036 4 1212 tcctggccatcctgatcaac 235 H. sapiens 646 231037 4 1062 gcttcttcagcctctacctg 236 H. sapiens 647 231038 4 559cttcgtgttcaagagatgcg 237 H. sapiens 648 231039 4 543acaaagtgcaacaccgcttc 238 H. sapiens 649 231041 4 1026 acaacctgctgggcctggcc 240 H. sapiens 650 231042 4 1070 agcctctacctgggcatcgg 241 H. sapiens 651 231043 4 496caataccacggccaacatct 242 H. sapiens 652 231044 4 399accagtgtcaccacaacctg 243 H. sapiens 653 231046 4 392tacggtgaccagtgtcacca 245 H. sapiens 654 231047 4 402agtgtcaccacaacctgagc 246 H. sapiens 655 110287 4 533ccttggcaccacaaagtgca 247 H. sapiens 656 231048 4 689tacagcagcttccaggtgat 248 H. sapiens 657 231049 4 956gccgcggtgttcatgcaata 249 H. sapiens 658 231051 4 555accgcttcgtgttcaagaga 251 H. sapiens 659 231052 4 553acaccgcttcgtgttcaaga 252 H. sapiens 660 231053 4 1027 caacctgctgggcctggcca 253 H. sapiens 661 231054 4 871caggacccgctacagccaga 254 H. sapiens 662 231055 4 498ataccacggccaacatctcc 255 H. sapiens 663 231056 4 259cagctagctgcccagaggca 256 H. sapiens 664 231057 4 1058 aggagcttcttcagcctcta 257 H. sapiens 665 231058 4 348cctccgctcaggtgatggac 258 H. sapiens 666 231061 4 953gtggccgcggtgttcatgca 261 H. sapiens 667 231062 4 1024 gcacaacctgctgggcctgg 262 H. sapiens 668 231063 4 1061 agcttcttcagcctctacct 263 H. sapiens 669 231064 4 1169 aatgacaacatgggcttctg 264 H. sapiens 670 231066 4 1021 cctgcacaacctgctgggcc 266 H. sapiens 671 231067 4 400ccagtgtcaccacaacctga 267 H. sapiens 672 231068 4 1165 cagcaatgacaacatgggct 268 H. sapiens 673 231069 4 363tggacttcctgtttgagaag 269 H. sapiens 674 231070 4 550gcaacaccgcttcgtgttca 270 H. sapiens 675 231071 4 367cttcctgtttgagaagtgga 271 H. sapiens 676 231073 4 1071 gcctctacctgggcatcggc 273 H. sapiens 677 231075 4 349ctccgctcaggtgatggact 275 H. sapiens 678 231077 4 463cgacaagtattcctgctggc 278 H. sapiens 679 231078 4 320ctgctgctggcctgccagcc 279 H. sapiens 680 231079 4 1183 cttctggtggatcctgcggt 280 H. sapiens 681 231080 4 862tgggctgctcaggacccgct 281 H. sapiens 682 231081 4 565gttcaagagatgcgggcccg 282 H. sapiens 683 231083 4 1177 catgggcttctggtggatcc 284 H. sapiens 684 231085 4 1184 ttctggtggatcctgcggtt 286 H. sapiens 685 231086 4 410cacaacctgagcctgctgcc 287 H. sapiens 686 231087 4 495ccaataccacggccaacatc 288 H. sapiens 687 231090 4 688gtacagcagcttccaggtga 291 H. sapiens 688 231092 4 863gggctgctcaggacccgcta 293 H. sapiens 689 231096 4 694cagcttccaggtgatgtaca 297 H. sapiens 690 231097 4 494gccaataccacggccaacat 298 H. sapiens 691 110307 4 1178 atgggcttctggtggatcct 300 H. sapiens 692 231099 4 1207 cgtcttcctggccatcctga 301 H. sapiens 693 231100 4 352cgctcaggtgatggacttcc 302 H. sapiens 694 231101 4 261gctagctgcccagaggcatg 303 H. sapiens 695 231102 4 561tcgtgttcaagagatgcggg 304 H. sapiens 696 231103 4 323ctgctggcctgccagccaca 305 H. sapiens 697 231104 4 324tgctggcctgccagccacag 306 H. sapiens 698 231105 4 1179 tgggcttctggtggatcctg 307 H. sapiens 699 231107 4 1289 atgcaccacacagactacaa 309 H. sapiens 700 231108 4 322gctgctggcctgccagccac 310 H. sapiens 701 231109 4 406tcaccacaacctgagcctgc 311 H. sapiens 702 231110 4 870tcaggacccgctacagccag 312 H. sapiens 703 231112 4 464gacaagtattcctgctggcc 314 H. sapiens 704 231114 4 1060 gagcttcttcagcctctacc 316 H. sapiens 705 231115 4 1422 tcttcttcgacctcttcctc 317 H. sapiens 706 231118 4 542cacaaagtgcaacaccgctt 321 H. sapiens 707 231119 4 456gaaccttcgacaagtattcc 322 H. sapiens 708 231122 4 404tgtcaccacaacctgagcct 325 H. sapiens 709 231123 4 538gcaccacaaagtgcaacacc 326 H. sapiens 710 231126 4 954tggccgcggtgttcatgcaa 329 H. sapiens 711 231127 4 684agatgtacagcagcttccag 330 H. sapiens 712 231129 4 1214 ctggccatcctgatcaactt 332 H. sapiens 713 231130 4 1023 tgcacaacctgctgggcctg 333 H. sapiens 714 231133 4 554caccgcttcgtgttcaagag 336 H. sapiens 715 231135 4 499taccacggccaacatctcct 338 H. sapiens 716 231136 4 1164 ccagcaatgacaacatgggc 339 H. sapiens 717 231138 4 1064 ttcttcagcctctacctggg 341 H. sapiens 718 231139 4 1163 accagcaatgacaacatggg 342 H. sapiens 719 231140 4 547agtgcaacaccgcttcgtgt 343 H. sapiens 720 231141 4 408accacaacctgagcctgctg 344 H. sapiens 721 231143 4 1020 acctgcacaacctgctgggc 346 H. sapiens 722 231145 4 562cgtgttcaagagatgcgggc 348 H. sapiens 723 231146 4 1418 aagctcttcttcgacctctt 349 H. sapiens 724 231148 4 557cgcttcgtgttcaagagatg 351 H. sapiens 725 231150 4 1155 agtgctggaccagcaatgac 353 H. sapiens 726 231151 4 566ttcaagagatgcgggcccga 354 H. sapiens 727 231152 4 721ctacagcctgtccctggggg 355 H. sapiens 728 110306 4 1162 gaccagcaatgacaacatgg 356 H. sapiens 729 231154 4 549tgcaacaccgcttcgtgttc 358 H. sapiens 730 231155 4 1159 ctggaccagcaatgacaaca 360 H. sapiens 731 231156 4 457aaccttcgacaagtattcct 361 H. sapiens 732 231160 4 546aagtgcaacaccgcttcgtg 365 H. sapiens 733 231162 4 260agctagctgcccagaggcat 367 H. sapiens 734 231163 4 690acagcagcttccaggtgatg 368 H. sapiens 735 231165 4 558gcttcgtgttcaagagatgc 370 H. sapiens 736 231166 4 958cgcggtgttcatgcaatatg 371 H. sapiens 737 231168 4 867tgctcaggacccgctacagc 373 H. sapiens 738 231169 4 865gctgctcaggacccgctaca 374 H. sapiens 739 231171 4 692agcagcttccaggtgatgta 376 H. sapiens 740 231172 4 1181 ggcttctggtggatcctgcg 377 H. sapiens 741 231174 4 1057 gaggagcttcttcagcctct 379 H. sapiens 742 231175 4 1211 ttcctggccatcctgatcaa 380 H. sapiens 743 231176 4 541ccacaaagtgcaacaccgct 381 H. sapiens 744 231177 4 319gctgctgctggcctgccagc 382 H. sapiens 745 231178 4 545aaagtgcaacaccgcttcgt 383 H. sapiens 746 231179 4 952tgtggccgcggtgttcatgc 384 H. sapiens 747 231181 4 1180 gggcttctggtggatcctgc 386 H. sapiens 748 231183 4 722tacagcctgtccctgggggc 388 H. sapiens 749 231185 4 957ccgcggtgttcatgcaatat 390 H. sapiens 750 231186 4 459ccttcgacaagtattcctgc 391 H. sapiens 751 110293 4 693gcagcttccaggtgatgtac 392 H. sapiens 752 231187 4 1153 ccagtgctggaccagcaatg 393 H. sapiens 753 110319 4 551caacaccgcttcgtgttcaa 396 H. sapiens 754 231190 4 1171 tgacaacatgggcttctggt 397 H. sapiens 755  63771 11  138caacctctgccagatgtggg 404 M. musculus 756  63777 11  274gtttgagaagtggaagctct 406 M. musculus 757  63778 11  284tggaagctctatagtgacca 407 M. musculus 758  63780 11  308caccacaacctaagcctgct 408 M. musculus 759  63784 11  403cactgccaacatttcctgcc 409 M. musculus 760  63787 11  464gtgttcaagaggtgtgggcc 410 M. musculus 761  63789 11  558agatcgaggtccagaagggg 411 M. musculus 762  63792 11  619gggctacagtctgtccctgg 412 M. musculus 763  63793 11  670gggcctcaggaagctgcact 413 M. musculus 764  63802 11  1019 ccctgggtggtggtcaagtg 414 M. musculus 765  63806 11  1058 tgctggaccagcaatgacaa 416 M. musculus 766  63808 11  1079 atgggattctggtggatcct 417 M. musculus 767  63811 11  1220 gccaggtccacgctgaccct 418 M. musculus 768  95505 400    14gcaacctgaggagaggtgca 424 M. musculus 769  95506 400    25agaggtgcacacactctgag 425 M. musculus 770  95507 400    30tgcacacactctgaggacct 426 M. musculus 771  95508 400    48ctaggtgtgcaacctctgcc 427 M. musculus 772  95509 400    80tggctacccagaggcatgcc 428 M. musculus 773  95511 400   192tgtttgagaagtggaagctc 430 M. musculus 774  95512 400   251ccacctactgagctggtctg 431 M. musculus 775  95513 400   291actcctgctggcctgacacc 432 M. musculus 776  95514 400   359tgccacaaagtgcagcaccg 433 M. musculus 777  95515 400   371cagcaccgcctagtgttcaa 434 M. musculus 778  95516 400   410cagtgggttcgagggccacg 435 M. musculus 779  95517 400   545agtctgtccctgggggcctt 436 M. musculus 780  95518 400   572gcgctggtcatcctgctggg 437 M. musculus 781  95519 400   582tcctgctgggcctcaggaag 438 M. musculus 782  95520 400   650gtgctcaaggctggctctgt 439 M. musculus 783  95521 400   764ggctgcagagtggccacagt 440 M. musculus 784  95522 400   775ggccacagtgatcatgcagt 441 M. musculus 785  95523 400   785atcatgcagtacggcatcat 442 M. musculus 786  95524 400   836gtgtacctgtacagcctgct 443 M. musculus 787  95525 400   974cagtgctggaccagcaatga 444 M. musculus 788  95527 400   1079 cttcttgtggccaagctgcg 446 M. musculus 789  95529 400   1100 gcccatcagatgcactatgc 448 M. musculus 790  95531 400   1256 ttcctcagctccttccaggg 450 M. musculus 791  95532 400   1292 ctctactgtttcctcaacaa 451 M. musculus 792  95533 400   1348 atggcaagaaggcaaagctc 452 M. musculus 793  95534 400   1360 caaagctcttcaggaggaaa 453 M. musculus 794  95535 400   1388 agcagccatggcagccacat 454 M. musculus 795  95536 400   1435 tccctgtgagaaacttcagc 455 M. musculus 796  95537 400   1450 tcagcttatgagtgcaggca 456 M. musculus 797  95538 400   1470 gcagcagtgggactggctgt 457 M. musculus 798  95539 400   1512 tggccagtagtctcccaagg 458 M. musculus 799  95540 400   1544 cccacctgaatctccacttg 459 M. musculus 800  95542 400   1575 ggttgtgttcaagaaagggc 461 M. musculus 801  95543 400   1600 aggacaacccagagccagat 462 M. musculus 802  95544 400   1610 agagccagatgcccggccaa 463 M. musculus 803  95545 400   1620 gcccggccaaggttgaagag 464 M. musculus 804  95546 400   1646 cagcaagacagcagcttgta 465 M. musculus 805  95547 400   1687 gtcctagcctggcacaggcc 466 M. musculus 806  95548 400   1724 gttggatatgatggagaagc 467 M. musculus 807  95549 400   1750 atctatgaactctgagtgtt 468 M. musculus 808  95550 400   1756 gaactctgagtgttcccatg 469 M. musculus 809  95551 400   1795 tacccagatatgtccttcag 470 M. musculus 810  95552 401   3953 cttggtctcgcctttgttac 471 M. musculus 811  95554 401   7321 accttcacagagctggtctg 473 M. musculus 812  95555 401   7505 ccatgcccagtgcagcaccg 474 M. musculus 813  95557 401   8766 gtcaggttgcccatccttgg 476 M. musculus 814  95560 11   10tcatgaggccttgggcttgg 479 M. musculus 815  95561 11   85ctggtcctctgcagcctgag 480 M. musculus 816

As these “preferred target segments” have been found by experimentationto be open to, and accessible for, hybridization with the antisensecompounds of the present invention, one of skill in the art willrecognize or be able to ascertain, using no more than routineexperimentation, further embodiments of the invention that encompassother compounds that specifically hybridize to these preferred targetsegments and consequently inhibit the expression of glucagon receptor.

According to the present invention, antisense compounds includeantisense oligomeric compounds, antisense oligonucleotides, ribozymes,external guide sequence (EGS) oligonucleotides, alternate splicers,primers, probes, and other short oligomeric compounds which hybridize toat least a portion of the target nucleic acid.

Example 17 Western Blot Analysis of Glucagon Receptor Protein Levels

Western blot analysis (immunoblot analysis) is carried out usingstandard methods. Cells are harvested 16-20 h after oligonucleotidetreatment, washed once with PBS, suspended in Laemmli buffer (100ul/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gelsare run for 1.5 hours at 150 V, and transferred to membrane for westernblotting. Appropriate primary antibody directed to glucagon receptor isused, with a radiolabeled or fluorescently labeled secondary antibodydirected against the primary antibody species. Bands are visualizedusing a PHOSPHORIMAGER™ (Molecular Dynamics, Sunnyvale Calif.).

Example 18 Effects of Antisense Inhibition of Glucagon Receptor in Miceon Plasma Glucose Levels and Glucagon Receptor mRNA Reduction: LeanAnimals, db/db Mice and ob/ob Mice

In accordance with the present invention, two antisense oligonucleotidestargeted to the mouse glucagon receptor, ISIS 148359 (agcaggcttaggttgtggtg, SEQ ID NO: 408) and ISIS 180475 (gagctttgcc ttcttgccat, SEQID NO: 452), were evaluated for therapeutic efficacy in art-acceptedmouse models of obesity and diabetes. Ob/ob mice have mutations in theleptin gene and are leptin-deficient, while db/db mice have mutations inthe leptin receptor gene. The two strains exhibit obesity and diabetesstrongly resembling Type 2 diabetes in humans. Tsang, S. H., 1998, P & SMedical Review, Vol. 5, No. 1.

Db/db and ob/ob mice were evaluated over the course of 4 weeks for theeffects of ISIS 148359 and ISIS 180475 on serum glucose levels andglucagon receptor mRNA levels, while normoglycemic mice were evaluatedfor 2 weeks. Control animals received saline treatment (50 mg/kg). Thenormoglycemic mice were dosed subcutaneously twice a week for 2 weekswith 50 mg/kg of ISIS 148359, ISIS 180475 or saline. The db/db and ob/obmice were dosed subcutaneously twice a week for 6 weeks with 25 mg/kg ofISIS 148359, ISIS 180475, saline, the positive control oligonucleotideISIS 116847 (ctgctagcc tctggatttga, SEQ ID NO: 817) or the negativecontrol oligonucleotide ISIS 141923 (ccttccctga aggttcctcc, SEQ ID NO:818). The mice were monitored weekly for fed or fasted plasma glucoselevels (fasted glucose measured 16 hr after last feeding) and upontermination of the experiment the level of glucagon receptor mRNA in theliver was determined. The data are summarized in Table 4.

TABLE 4 Effects of ISIS 148359 and ISIS 180475 treatment on fed andfasting glucose levels and glucagon receptor mRNA levels innormoglycemic mice, db/db mice, and ob/ob mice Biological ISIS #Antisense Marker mice (time day of Oligonucleotides Controls Measuredcourse of study) treatment 148359 180475 saline 116847 141923 fed leanmice −6 221 216 210 N.D. N.D. plasma (2 week) 15 151 130 181 N.D. N.D.glucose db/db mice −1 294 295 296 295 304 mg/dL (4 weeks)  5 361 329 460355 408 12 375 303 510 303 425 26 314 222 495 354 493 ob/ob mice −1 338342 343 337 361 (4 weeks) 12 245 180 426 227 476 27 168 145 394 205 431fasted db/db mice 19 336 232 320 321 298 plasma (4 weeks) 29 254 150 302193 262 glucose ob/ob mice 19 205 132 317 167 245 mg/dL (4 weeks) 29 178117 322 189 340 glucagon lean mice end 82 93 0 N.D. N.D. receptor (2week) % mRNA db/db mice end 74 96 0  0  0 reduction (4 weeks) ob/ob miceend 86 97 0  10  0 (4 weeks)

These data demonstrate that the antisense oligonucleotides ISIS 148359and ISIS 180475 targeted to glucagon receptor mRNA are capable ofdecreasing levels of glucagon receptor mRNA in mouse liver. These datafurther demonstrate that reduction of glucagon receptor expression isaccompanied by a decrease in plasma glucose levels in normoglycemicmice, db/db mice and ob/ob mice. It is important to note that thetreated mice become normoglycemic and do not become hypoglycemic.Antisense inhibitors of glucagon receptor are thus believed to be usefultherapeutic modalities for treatment of hyperglycemia.

Example 19 Glucagon Receptor Antisense Oligonucleotides Lower PlasmaGlucose in ob/ob Diabetic Mice 4 Week Study

C57Bl/eOlaHsd-Lep_(ob) (ob/ob) male mice were purchased from Harlan(Indianapolis, Ind., USA). Animals were acclimated for one week prior tostudy initiation. Mice were housed five per cage in polycarbonate cageswith filter tops. Animals were maintained on a 12:12 hr light-dark cycle(lights on at 6:00 AM) at 21° C. All animals received de-ionized waterad libitum. ob/ob mice received Purina Diet 5015 ad libitum. Antisensecompounds were prepared in normal saline, and the solution wassterilized through a 0.2 μm filter. Animals were dosed with antisensecompound solutions or vehicle (saline) twice per week (separated by 3.5days) via subcutaneous injection. Before the initiation of each studyand once weekly during the study, blood was collected by tail clipwithout anesthesia into EDTA plasma tubes containing trasylol(Serologicals Proteins, Kankakee, Ill., USA) and dipeptidyl peptidase(DPP)-IV inhibitor (Linco Diagnostic Services, St. Charles, Mo., USA).Food intake and body weights were measured weekly. Plasma levels ofglucose and triglycerides were determined on the Hitachi 912 clinicalchemistry analyzer (Roche, Indianapolis, Ind., USA).

To test the efficacy of antisense inhibitors of glucagon receptor totreat hyperglycemia, 7-8 week-old ob/ob mice were dosed two times perweek with antisense inhibitors of glucagon receptor [ISIS 148359 (SEQ IDNO: 408) or ISIS 180475 (SEQ ID NO: 452)], a generic controloligonucleotide (ISIS 141923; SEQ ID NO: 818) whose sequence does notmatch any known transcripts in the mouse or rat genomes, a mismatcholigonucleotide (ISIS 298682; GCGATTTCCCGTTTTGACCT; SEQ ID NO: 819)whose sequence is identical to ISIS 180475 except for 7 internal bases,or saline twice a week (every 3.5 days) for 4 weeks. Alloligonucleotides were administered at 25 mg/kg. Data are the mean values(±SEM where shown) of 8 mice per treatment group. Plasma glucose levelsin all mice were approximately 330-370 mg/dl day zero. Whereashyperglycemia worsened over time in saline- and controloligonucleotide-treated ob/ob mice, animals treated with glucagonreceptor antisense compounds showed a dramatic reduction in plasmaglucose. At day 12, plasma glucose levels in ob/ob mice treated withcontrol oligonucleotide (ISIS 141923) and saline were approximately 472and 425 mg/dl, respectively. Plasma glucose levels in mice treated withantisense oligonucleotides ISIS 148359 and ISIS 180475 were 240 and 180mg/dl, respectively. At day 27, plasma glucose levels in ob/ob micetreated with control oligonucleotide (ISIS 141923) and saline wereapproximately 435 and 390 mg/dl, respectively. Plasma glucose levels inmice treated with antisense oligonucleotides ISIS 148359 and ISIS 180475were 165 and 130 mg/dl, respectively. The latter is in the normal range.

A separate study, also using ob/ob mice (as well as db/db mice, leanmice, ZDF rats and lean rats) was also performed in which animals werealso dosed subcutaneously every 3.5 days for a total of 9 doses ofglucagon receptor antisense compound ISIS 180475 and one or morecontrols (unrelated control oligonucleotide ISIS 141923, mismatchcontrol oligonucleotide ISIS 298682, and/or saline). The results of thisstudy are shown in Table 5.

At the end of the 4-week treatment period, liver glucagon receptor mRNAwas measured (normalized to total RNA in the same samples usingRibogreen) and was found to be reduced by 85-95%. Data are mean valuesof four mice per treatment group (P<0.05 using Student's t-test).

TABLE 5 Effects of antisense inhibition of glucagon receptor in rodentsPlasma Plasma Plasma Plasma Glucose Triglycerides Insulin Glucagon BodyWeight (g) (mg/dl) (mg/dl) (ng/ml) (pg/ml) ob/ob mice Saline 56.5 ± 1.5 564 ± 118 163 ± 25 35.9 ± 13.8 n.d. ISIS 180475 54.1 ± 1.6 122 ± 6* 129± 7  19.8 ± 9.5  n.d. db/db mice ISIS 141923 46.0 ± 0.7 571 ± 29 412 ±33 n.d. 117 ± 12  ISIS 298682 43.9 ± 1.3 577 ± 65 448 ± 40 n.d. 131 ±20  ISIS 180475 45.6 ± 0.6  241 ± 37*  121 ± 12* n.d. 3765 ± 952*db^(+/?) lean mice ISIS 141923 28.0 ± 1.0 196 ± 12 121 ± 7  n.d. 80 ± 1 ISIS 180475 27.6 ± 1.0 164 ± 4*  83 ± 6* n.d. 362 ± 40* ZDF rats ISIS141923 403 ± 12 417 ± 38  640 ± 105 5.0 ± 1.9 136 ± 7  ISIS 180475 404 ±8   143 ± 15*  250 ± 25* 4.4 ± 0.5 548 ± 20* SD lean rats Saline 344 ±4  116 ± 3  106 ± 26 2.7 ± 0.3 56 ± 11 ISIS 180475 327 ± 5  104 ± 7  139± 58  1.3 ± 0.2*  855 ± 122* *P < 0.05. n.d., not determined

Example 20 Glucagon Receptor Antisense Oligonucleotides Lower PlasmaGlucose in db/db Diabetic Mice 4 Week Study

C57Bl/KsOlaHsd-Lep_(db) (db/db) and lean (db^(+/+)) male mice werepurchased from Harlan (Indianapolis, Ind., USA). Animals were acclimatedfor one week prior to study initiation. Mice were housed five per cagein polycarbonate cages with filter tops. Animals were maintained on a12:12 hr light-dark cycle (lights on at 6:00 AM) at 21° C. All animalsreceived de-ionized water ad libitum. db/db mice received Purina Diet5008 ad libitum. Antisense compounds were prepared in normal saline, andthe solution was sterilized through a 0.2 μm filter. Animals were dosedwith antisense compound solutions or vehicle (saline) twice per week(separated by 3.5 days) via subcutaneous injection. Before theinitiation of each study and once weekly during the study, blood wascollected by tail clip without anesthesia into EDTA plasma tubescontaining trasylol (Serologicals Proteins, Kankakee, Ill., USA) anddipeptidyl peptidase (DPP)-IV inhibitor (Linco Diagnostic Services, St.Charles, Mo., USA). Food intake and body weights were measured weekly.Plasma levels of glucose and triglycerides were determined on theHitachi 912 clinical chemistry analyzer (Roche, Indianapolis, Ind., USA.

To test the efficacy of antisense inhibitors of glucagon receptor totreat hyperglycemia, 7-8 week-old db/db mice were dosed two times perweek with antisense inhibitors of glucagon receptor [ISIS 148359 (SEQ IDNO: 408) or ISIS 180475 (SEQ ID NO: 452)], a generic controloligonucleotide (ISIS 141923) whose sequence does not match any knowntranscripts in the mouse or rat genomes, a mismatch oligonucleotide(ISIS 298682; SEQ ID NO: 819) whose sequence is identical to ISIS 180475except for 7 internal bases, or saline for 4 weeks.

Glucose lowering efficacy and target reduction in db/db mice undergoingglucagon receptor antisense treatment were similar to those observed insimilarly treated ob/ob mice; furthermore, plasma triglycerides werelowered from 412±33 to 121±12 mg/dl following glucagon receptorantisense treatment (Table 5). These results in db/db mice are similarto those reported in preliminary studies testing glucagon receptorantisense compound ISIS 148359 for 3 weeks [Osborne et al., 2003,Diabetes 52, A129 (abstract)].

Example 21 Glucagon Receptor Antisense Oligonucleotides Lower PlasmaGlucose in ZDF Rats

ZDF/GmiCrl-fa/fa (ZDF) male rats were purchased from Charles RiverLaboratories (Wilmington, Mass., USA). Animals were acclimated for oneweek prior to study initiation. Rats were housed one per cage inpolycarbonate cages with filter tops. Animals were maintained on a 12:12hr light-dark cycle (lights on at 6:00 AM) at 21° C. All animalsreceived de-ionized water ad libitum. ZDF rats received Purina Diet 5008ad libitum. Antisense compounds were prepared in normal saline, and thesolution was sterilized through a 0.2 μm filter. Seven-week old animalswere dosed with antisense compound solutions or vehicle (saline) twiceper week (separated by 3.5 days) via subcutaneous injection, for a totalof 9 doses (last treatment on day 28), followed by a washout period ofequal duration. Oligonucleotide concentration was 25 mg/kg of eitherglucagon receptor antisense oligonucleotide ISIS 180475 (SEQ ID NO: 452)or negative control oligonucleotide ISIS 141923 (SEQ ID NO: 818). Beforethe initiation of each study and once weekly during the study, blood wascollected by tail clip without anesthesia into EDTA plasma tubescontaining trasylol (Serologicals Proteins, Kankakee, Ill., USA) anddipeptidyl peptidase (DPP)-IV inhibitor (Linco Diagnostic Services, St.Charles, Mo., USA). Food intake and body weights were measured weekly.Glucagon receptor mRNA (target) was measured by real-time quantitativeRT-PCR from livers of five animals removed from the study at each timepoint. Rat 36B4 ribosomal phosphoprotein mRNA (“18S RNA”) was measuredand used to normalize RNA input. Data are the mean values of five ratsper treatment group. In overall comparisons during the treatment period,target reduction by glucagon receptor antisense compound ISIS 180475 wassignificantly different when compared to control oligonucleotide-treatedanimals (P<0.05 adjusted using the Tukey method). Liver glucagonreceptor mRNA decreased dramatically to 50% of controls within 24 hoursafter the first dose of ISIS 180475 and to 30% of controls 48 hrfollowing the seventh dose.

For non-fasted plasma glucose levels, rats were treated as describedabove. Data are the mean values of five rats per treatment group. Inoverall comparisons during the treatment period, glucose lowering by theglucagon receptor antisense compound ISIS 180475 showed significantdifference when compared to control oligonucleotide-treated animals.(P<0.05 adjusted using the Tukey method). The drop in plasma glucoseparalleled the drop in glucagon receptor mRNA levels; there was asignificant drop in plasma glucose within 48 hours after the initialglucagon receptor antisense dose. After 9 doses, the controloligonucleotide (ISIS 141923) treated rats had plasma glucose levelsaveraging approximately 417 mg/dl and antisense (ISIS 180475) treatedrats had plasma glucose levels averaging approximately 143 mg/dl.

During the washout phase, hyperglycemia and glucagon receptor expressionin liver began to rebound within 10 days, but even one month after thefinal dose, efficacy was still observed as plasma glucose and targetmRNA levels in washout animals remained below pre-treatment levels.Glucose lowering achieved by the twice per week dosing schedule and thegradula rebound of glucagon receptor mRNA during the washout period areboth consistent with the extended half lives of 2′-methoxyethoxymodified phosphorothioate oligonucleotides (typically ranging from 9 to19 days according to published reports).

Non-fasted plasma insulin levels were also determined for rats treatedas described above. Data are the mean values of five rats per treatmentgroup. No significant changes were observed during the treatment period;however, individual comparisons between glucagon receptor antisense andcontrol oligonucleotide treated animals on day 38 and 56 (washoutperiod) were significant (P<0.05). Plasma insulin levels declined duringthe treatment phase in both control oligonucleotide andantisense-treated animals. During the washout phase of the controloligonucleotide treated group, insulin levels continued to decline ashyperglycemia progressed. This result is expected since beta-cellfailure typically occurs in ZDF rats between 8 and 12 weeks of age.Interestingly, the mild elevation of glucose in glucagon receptorantisense-treated animals during the washout period resulted in a robustrise in plasma insulin to levels nearly as high as at start of study.This is consistent with evidence of preserved beta-cell function.

Example 22 Glucagon Receptor Antisense Oligonucleotides do not CauseHyperglycemia or Hypoglycemia, in Spite of Hyperglucagonemia

In addition to effects on blood glucose, treatment with the antisenseinhibitor of glucagon receptor (ISIS 180475; SEQ ID NO: 452) resulted inmarked (and reversible) hyperglucagonemia in both normal and diabeticrodents (Table 5). This level of hyperglucagonemia is similar to thatobserved in glucagon receptor knockout mice (Parker et al., 2002,Biochem Biophys Res Commun. 290, 839-843; Gelling et al., 2003, Proc.Natl. Acad. Sci. USA., 100, 1438-1443. Because of these high levels ofserum glucagon, it was important to determine whether the antisenseinhibitors of glucagon receptor might induce hyperglycemia, particularlyas hepatic glucagon receptor levels gradually return to normal followingtreatment withdrawal. It is therefore significant that at no time duringthe treatment or washout periods did animals with hyperglucagonemiaexhibit hyperglycemia. In fact, glucagon receptor antisense-treatedanimals showed a moderate decrease in fed plasma glucose at all timepoints tested.

It was also confirmed that antisense treatment also did not causehypoglycemia. After 4 weeks of antisense treatment, by which timemaximum reduction of glucagon receptor expression had been achieved,db^(+/?) lean mice were subjected to periods of fasting of up to 24hours. Although the glucagon receptor antisense-treated mice displayed a10-30% reduction in plasma glucose, at no time did the animals reachadverse levels of hypoglycemia. This is in contrast to Gcgr knockoutmice, which become hypoglycemic during periods of fasting. Gelling etal., 2003, Proc Natl. Acad. Sci. U.S.A. 100, 1438-1443.

Example 23 Glucagon Receptor mRNA is Reduced in Islets ofAntisense-Treated db/db Mice

Pancreatic islets were isolated from 12-week-old male db/db mice (n=5-6per treatment group) that had been treated twice per week (every 3.5days) by subcutaneous injection with saline or glucagon receptorantisense oligonucleotide ISIS 180475 (SEQ ID NO: 452) at 25 mg/kg for atotal of 9 doses. Mice were sacrificed by cervical dislocation. Thecommon bile duct was cannulated with a 27-gauge needle and the pancreaswas distended with 3 ml of Hank's buffer (Sigma, Taufkirchen, Germany)containing 2% bovine serum albumin (Applichem, Darmstadt, Germany) and 1mg/ml collagenase (Serva, Heidelberg, Germany). Subsequently, thepancreas was removed and digested in Hank's buffer at 37° C. Islets werepurified on a Histopaque-1077™ (Sigma) gradient for 15 min at 750×g.Islets were cultured overnight in RPMI-1640 medium containing 10% FBS,100 U/ml penicillin, and 100 μg/ml streptomycin (Invitrogen, Karlsruhe,Germany). 200 islets from 3 individuals were pooled to give one samplefor RNA extraction. Real-time quantitative RT-PCR was used to profilegene expression. Islet glucagon receptor mRNA levels were decreased byapproximately 75% in antisense-treated animals compared tosaline-treated controls. It should be noted that, in addition topharmacologic effect of the antisense compound, a compensatory responseto hyperglucagonemia or the increased alpha-cell populations in treatedanimals could contribute to the results observed.

Example 24 Glucagon Receptor Antisense Oligonucleotides Decrease theNumber of Functional Glucagon Receptors

To assess whether the reduction in glucagon receptor mRNA correlateswith a reduction in functional glucagon receptor number, a homologouscompetition assay was performed using hepatocyte membranes prepared frommice treated with control or glucagon receptor antisense compounds.¹²⁵I-glucagon binding was effectively competed by increasingconcentrations of unlabeled glucagon in control membrane samples. 15-20μg of membrane from control oligonucleotide- or glucagon receptoroligonucleotide (ISIS 180475; SEQ ID NO: 452)-treated db/db mice wereincubated with 0.1 nM ¹²⁵I-glucagon (2000 Ci/mmol, PerkinElmer, Boston,Mass., USA) and the indicated concentrations of unlabeled glucagon (EliLilly and Company, Indianapolis, Ind., USA) in buffer containing 50 mMHepes, 1 mM MgCl₂, 5 mM EGTA, 0.005% Tween 20, 0.1% BSA, and EDTA-freeprotease inhibitor cocktail (Roche). Assays were performed under steadystate conditions in the presence of excess labeled ligand on 96-wellMultiScreen-HV 0.45 μm filter plates (Millipore, Bedford, Mass., USA).Following incubation for 2 hrs at room temperature, plates were rapidlywashed by filtration with ice-cold buffer (20 mM Tris, pH 7.4) and driedfor 45 min at 50° C. Following the addition of Optiphase Supermix(PerkinElmer), plates were counted on a Wallac Microbeta scintillationcounter. Data analyses were performed using GraphPad Prism software andexpressed as mean+/−SEM. Data obtained for samples from animals treatedwith glucagon receptor oligonucleotide neared the limits of detectionfor the assay and curve-fitting parameters. In order to derive anumerical value for apparent Bmax, the Kd was fixed at the average value(0.69+/−0.2 nM) obtained from the samples from the controlantisense-treated animals.

Functional GCGR expression was found to be decreased approximately 85%by glucagon receptor antisense treatment and is in accord withquantitative RT-PCR results.

Example 25 Antisense Inhibitors of Human and Monkey Glucagon ReceptorDose Response

Based on the screen in Example 15 above, a subset of human glucagonreceptor antisense oligonucleotides were chosen for further study.Dose-response studies were conducted for ISIS 315186, 310457, 315324,315278, 315181, 315297, 315163 and 310456 in both human HepG2 cellcultures and in cynomolgus monkey primary hepatocytes. These sixcompounds are homologous to both human and cynomolgus monkey glucagonreceptor nucleic acid targets. The universal control ISIS 29848(NNNNNNNNNNNNNNNNNNNN; SEQ ID NO: 820, where N is an equimolar mixtureof A, C, G and T, a chimeric 2′ MOE gapmer with a phosphorothioatebackbone and with MOEs at positions 1-5 & 16-20) was used as negativecontrol.

The human hepatoblastoma cell line HepG2 was obtained from the AmericanType Culture Collection (Manassas, Va.). HepG2 cells are routinelycultured in Eagle's MEM supplemented with 10% fetal calf serum,non-essential amino acids, and 1 mM sodium pyruvate (Gibco/LifeTechnologies, Gaithersburg, Md.). Cells are routinely passaged bytrypsinization and dilution when they reach 90% confluence.

Primary cynomolgus monkey hepatocytes were obtained from CellzDirect(Los Angeles) and plated onto collagen-coated 24-well plates (Costar) ata density of 75,000 cells/well. The culturing medium for thesehepatocytes was William's E media (Invitrogen) supplemented with 10% FBS(Invitrogen). Cells were allowed to attach overnight and were thentreated with oligonucleotide-Lipofectin mixture for 4 hours. Theoligonucleotide-Lipofectin mixture was washed off and then cells wereincubated in normal medium.

Cells were treated with oligonucleotide for 20 hours at doses of 1, 5,10, 25, 50, 100 nM for HepG2 cells and 5, 10, 25, 50, 100 and 200 nM forprimary monkey hepatocytes (n=3). RNA was analyzed by RT-PCR todetermine percent inhibition of glucagon receptor expression compared tocontrol (ISIS 29848), at each oligonucleotide concentration. The resultswere plotted to give the IC50, the dose of oligonucleotide which resultsin 50% reduction of glucagon receptor mRNA levels. Results are shown inTable 6.

TABLE 6 IC50s of glucagon receptor antisense oligonucleotides in humanHepG2 cells and in cynomolgus monkey primary hepatocytes (in nM) SEQ IDIC50 in human IC50 in monkey ISIS # NO: HepG2 cells (nM) hepatocytes(nM) 315186 254 5 30 310457 184 7 10 315324 392 6 19 315278 346 8 25315181 249 9 32 315297 365 11 11 315163 231 19 37 310456 183 25 20Based on these results, three compounds (ISIS 315297, 310457 and 315163)were chosen for study in monkeys.

Example 26 Dose-Ranging Study of Antisense Inhibition of GlucagonReceptor Expression in Cynomolgus Monkeys

A monkey study was performed at SNBL USA, Ltd., Everett, Wash. Fortymature (6-8 years old) male Macaca fascicularis (purpose-bred cynomolgusmonkeys) weighing approximately 5-10 kg at the initiation of dosing wereused. The animals were individually housed in primary climate-controlledenclosures conforming to the Animal Welfare Act. Animals were offeredPurina Mills Laboratory Profiled Fiber Plus® Monkey Diet (AnimalSpecialties, Hubbard, Oreg.). Occasional fresh fruit and vegetabletreats were also offered. Fresh drinking water was available to allanimals, ad libitum. Before fasting measurements, food was removed fromenclosures between 1630 and 1700 on the afternoon before the scheduledblood draw. After the animals were fasted for at least 16 hours, bloodsamples for plasma analysis were collected BEFORE dosing or feeding. Forfasting analysis, approximately 2.3-2.5 mL of blood was drawn from aperipheral vein. Approximately 1.8 to 2.0 mL was be deposited into aK2-EDTA tube containing DPP-IV inhibitor at 10 μL/mL of blood andtrasylol at 250 KIU/mL of blood. Approximately 0.5 mL was deposited intoa lithium heparin tube. Once the blood was been deposited into the EDTAplus additives tube, it was inverted to mix and placed on ice within 5minutes. The blood in the lithium heparin tube was also placed on icewithin 5 minutes. Blood samples were centrifuged (2000×g, 15 minutes at4 to 8° C.) to obtain plasma within 30 minutes of sample collection. Theplasma was frozen at or below −70° C. Samples were shipped on dry icevia overnight courier as described below for subsequent analysis. Fornon-fasted analysis, the animals were given their AM feeding (between0830 and 0930) on the day of the blood draw. Ninety minutes afterfeeding, blood was drawn. The number of biscuits remaining were countedat the time of the blood draw. Samples are prepared and shipped asabove.

Monkeys were dosed subcutaneously for 10 weeks with ISIS 315297, ISIS310457 or ISIS 315163 at three concentrations. In week 1,oligonucleotides were given at 2.0, 5.0 and 20 mg/kg/dose (Day 1, 3 and5); in week 2 through 10, oligonucleotides were given at 1.0, 2.5 and 10mg/kg/dose (twice weekly starting at Day 8) The larger dose (6, 15 or 60mg/kg/week, i.e. 3 injections of 2, 5 or 20 mg/kg) was given in week 1in order to rapidly achieve the desired steady state oligonucleotideconcentration. In week 1, compounds were administered 3 times, everyother day; for weeks 2-10, compounds were administered twice per week,with at least 2 days between dosings.

Oligonucleotides were given by subcutaneous (SC) injection, usingvolumes of 0.1-0.3 ml/kg). For each dosing of each animal, theappropriate volume of the relevant ASO solution or of thecontrol/vehicle article was administered subcutaneously using a syringeand needle (27G). The total volume of the relevant dosing solution orthe control article was calculated on the basis of the animal's mostrecent body weight. Multiple injection sites on the upper back(intra-scapular region) of each monkey were employed. During acclimationthe skin of the upper back was be shaved and a clock-like grid (pointsat 12, 3, 6, and 9 o'clock) was tattooed on each animal. Injectionpoints were a minimum of 5 cm apart. The injection sites were rotated sothat each site was used for fourth dose, starting with 12 o'clock androtating clockwise. The needle was inserted away from the dot and angledso that the dose was deposited underneath the dot.

After the 10 week study (approx. 2 days after last dose), animals wereeuthanized and three 1 to 4 gram samples of liver tissue were removedand individually snap frozen over liquid nitrogen; alternatively,biopsies could be taken from living animals and frozen. Frozen tissueswere homogenized in 4M guanidinium isothiocyanate solution and subjectedto CsCl centrifugation (150,000×g for 16 hr at 18° C.). The supernatantwas removed and the RNA pellet was resuspended in water, following whichit was applied to RNEASY mini columns (Qiagen, Valencia Calif.). Afterpurification and quantitation, the tissues were subjected to RT-PCRanalysis as described in previous examples using the following primersand probe:

Forward primer ACTGCACCCGCAACGC (SEQ ID NO: 821) Reverse primerCACGGAGCTGGCCTTCAG (SEQ ID NO: 822) Probe ATCCACGCGAACCTGTTTGTGTCCTT(SEQ ID NO: 823)RNA amounts were normalized to 18S RNA levels in the tissue. Results areshown in Table 7, as percent reduction in glucagon receptor mRNA inantisense-treated monkeys compared to saline-treated monkeys.

TABLE 7 Glucagon receptor mRNA reduction in monkey liver after treatmentwith antisense inhibitors of glucagon receptor - RT-PCR expt 1 SEQ ID %reduction % reduction % reduction ISIS # NO: at 2 mg/kg At 5 mg/kg at 20mg/kg 310457 184 17 31 64 315297 365 2 21 49 315163 231 22 18 47RNA analysis of the same tissue samples by RT-PCR was repeatedindependently using the same primer-probe set as above. Results areshown in Table 8 as percent reduction in glucagon receptor mRNA inantisense-treated monkeys compared to saline-treated monkeys.

TABLE 8 Glucagon receptor mRNA reduction in monkey liver after treatmentwith antisense inhibitors of glucagon receptor -RT-PCR expt 2 SEQ ID %reduction % reduction % reduction ISIS # NO: at 2 mg/kg At 5 mg/kg at 20mg/kg 310457 184 25 23 63 315297 365 18 21 56 315163 231 25 29 44The results obtained by RT-PCR were confirmed by Northern blot analysisaccording to standard methods (Example 14). The cDNA probe that was usedfor northern blots was a 900-base fragment of monkey GCGR generated byRT-PCR from cynomolgus monkey liver. Results are shown in Table 9.

TABLE 9 Glucagon receptor mRNA reduction in monkey liver after treatmentwith antisense inhibitors of glucagon receptor - Northern blot SEQ ID %reduction % reduction % reduction ISIS # NO: at 2 mg/kg At 5 mg/kg at 20mg/kg 310457 184 8 16 65 315297 365 0 10 38 315163 231 8 30 27

Blood glucose levels were measured in monkeys after treatment withantisense inhibitors of glucagon receptor. Glucose readings wereperformed using a drop of blood from the blood samples collected asabove and read on a One Touch Profile® (Lifescan Inc., a Johnson andJohnson Company). Because normoglycemic (nondiabetic) monkeys were usedin this study, no significant changes in blood glucose levels wereexpected or observed. At no point did animals become hypoglycemic afterantisense treatment.

Glucagon levels were measured in plasma of fasted monkeys before(baseline) and after treatment for 5 weeks or 10 weeks with antisenseinhibitors of glucagon receptor. Monkeys were anesthetized prior toblood collection to avoid artifacts due to stress. Glucagon levels weredetermined by radioimmunoassay, ELISA and/or Luminex immunoassay bycontract laboratory (Linco, St. Charles Mo.). Results are shown in Table10.

TABLE 10 Fasted glucagon levels in monkey liver after treatment withantisense inhibitors of glucagon receptor Glucagon Glucagon AntisenseGlucagon (pg/ml) (pg/ml) SEQ ID Dose (pg/ml) Week 5 Week 10 ISIS # NO:(mg/kg) Baseline fasted fasted Saline 155 ± 31 150 ± 19  250 ± 141310457 184 2  487 ± 123 278 ± 54 189 ± 36 5 211 ± 25 179 ± 55 169 ± 2620 308 ± 77  580 ± 247 1247 ± 451 315297 365 2 410 ± 94 140 ± 42 133 ±26 5 519 ± 58 193 ± 31 204 ± 30 20 375 ± 87 209 ± 23 276 ± 67 315163 2312 176 ± 42 152 ± 33 143 ± 26 5 262 ± 76 203 ± 74 225 ± 95 20 251 ± 40257 ± 76  421 ± 197

Glucagon-like peptide 1 (GLP-1) levels were measured in plasma of fastedmonkeys before (baseline) and after treatment for 5 weeks or 10 weekswith antisense inhibitors of glucagon receptor. Monkeys wereanesthetized prior to blood collection to avoid artifacts due to stress.GLP-1 levels were determined by radioimmunoassay, ELISA and/or Lumineximmunoassay by contract laboratory (Linco, St. Charles Mo.). Results areshown in Table 11.

TABLE 11 Fasted GLP-1 levels in monkey liver after treatment withantisense inhibitors of glucagon receptor Antisense GLP-1 GLP-1 (pM)GLP-1 (pM) SEQ ID Dose (pM) Week 5 Week 10 ISIS # NO: (mg/kg) Baselinefasted fasted Saline 4 ± 2 4 ± 1 5 ± 1 310457 184 2 4 ± 1 3 ± 1   3 ±.41 5 4 ± 1   4 ± .48 3 ± 1 20 8 ± 3 17 ± 6  30 ± 15 315297 365 2 3 ± 13 ± 1   3 ± .29 5 4 ± 1 4 ± 1 4 ± 2 20 11 ± 8  9 ± 4 7 ± 5 315163 231 24 ± 1 4 ± 1 4 ± 1 5 3 ± 1 5 ± 2 3 ± 1 20 2 ± 0   4 ± .48 5 ± 1

1. An antisense compound 12 to 50 nucleobases in length comprising atleast one of a modified internucleoside linkage, a high affinitymodified sugar or a modified nucleobase, wherein the compound targetsand specifically hybridizes with at least 90% complementarity withinnucleotides 532 to 558 of a nucleic acid molecule encoding a humanglucagon receptor having SEQ ID NO:4.
 2. The compound of claim 1 whichis 15 to 30 nucleobases in length.
 3. The compound of claim 1 which is20 nucleobases in length.
 4. The compound of claim 1 having at least95%. complementarity with a nucleic acid molecule encoding humanglucagon receptor having SEQ ID NO:
 4. 5. The compound of claim 1 havingat least one 2′-O-methoxyethyl sugar moiety.
 6. The compound of claim 1having at least one phosphorothioate internucleoside linkage.
 7. Thecompound of claim 1 having at least one 5-methylcytosine.
 8. Thecompound of claim 1 that is a pharmaceutically acceptable salt.
 9. Apharmaceutical composition comprising the compound of claim 1 and apharmaceutically acceptable diluent or carrier.
 10. The compound ofclaim 1 comprising at least one high affinity modified sugar moietyselected from the group consisting of a 2′-O-(2-methoxyethyl), a2′-O-methyl or a locked nucleic acid.
 11. The compound of claim 1wherein the compound is chimeric, comprising deoxynucleotides in a gapregion, at least one high affinity modified sugar in each of a firstwing region, and a second wing region, said wing regions flanking thegap region on the 5′ end and the 3′ end, respectively, and at least onephosphorothioate modified internucleoside linkage.
 12. The compound ofclaim 11 wherein the gap segment is ten deoxynucleotides in length, thefirst and second wing segments are each five nucleotides in length andcomprise five 2′-O-(2-methoxyethyl) nucleotides, and eachinternucleoside linkage in the chimeric oligonucleotide is aphosphorothioate.
 13. The compound of claim 1 wherein at least a portionof said compound hybridizes with RNA to form an oligonucleotide-RNAduplex.
 14. The compound of claim 11 further comprising at least one5-methylcytosine modified nucleobase.
 15. The compound of claim 1comprising a pharmaceutically acceptable salt.
 16. The compound of claim1, wherein the compound targets and specifically hybridizes withinnucleotides 532 to
 558. 17. A method of inhibiting the expression ofhuman glucagon receptor in cells or tissues comprising contacting saidcells or tissues with an antisense compound 12 to 50 nucleobases inlength comprising at least one of a modified internucleoside linkage, ahigh affinity modified sugar or a modified nucleobase, wherein thecompound is targeted to a nucleic acid molecule encoding human glucagonreceptor and wherein the compound specifically hybridizes with at least90% complementarity with nucleotides 532 to 558 of a nucleic acidmolecule having the sequence SEQ ID NO:4 and inhibits the expression ofthe human glucagon receptor.
 18. A method of treating or delaying theonset of a disease or condition associated with glucagon receptor in ahuman comprising administering to said human a therapeutically orprophylactically effective amount of an antisense compound 12 to 50nucleobases in length comprising at least one of a modifiedinternucleoside linkage, a high affinity modified sugar or a modifiednucleobase, wherein the compound is targeted to a nucleic acid moleculeencoding human glucagon receptor and wherein the compound specificallyhybridizes with at least 90% complementarity with nucleotides 532 to 558of a nucleic molecule having the sequence SEQ ID NO:4, and inhibits theexpression of the human glucagon receptor, thereby treating or delayingthe onset of the disease or condition.
 19. The method of claim 18,wherein the disease or condition is a metabolic disease or condition.20. The method of claim 19, wherein the metabolic disease or conditionis diabetes, obesity, hyperglycemia, primary hyperglucagonemia, insulindeficiency or insulin resistance.
 21. The method of claim 20 wherein thediabetes is Type 2 diabetes.
 22. The method of claim 19, wherein themetabolic disease or condition is associated with elevated blood glucoselevels, elevated blood triglyceride levels or elevated blood cholesterollevels.
 23. A method of decreasing blood glucose levels in a humancomprising administering to said human a therapeutically orprophylactically effective amount of an antisense compound 12 to 50nucleobases in length comprising at least one of a modifiedinternucleoside linkage, a high affinity modified sugar or a modifiednucleobase, wherein the compound is targeted to a nucleic acid moleculeencoding human glucagon receptor and wherein the compound specificallyhybridizes with at least 90% complementarity within nucleotides 532 to558 of a nucleic acid molecule having the sequence SEQ ID NO:4 andinhibits the expression of the human glucagon receptor.
 24. The methodof claim 23 wherein the blood glucose levels are plasma glucose levels.25. The method of claim 23 wherein the human suffers from diabetes orobesity.
 26. A method of preventing or delaying the onset of an increasein blood glucose levels in a human comprising administering to saidhuman a therapeutically or prophylactically effective amount of anantisense compound 12 to 50 nucleobases in length comprising at leastone of a modified internucleoside linkage, a high affinity modifiedsugar or a modified nucleobase, wherein the compound is targeted to anucleic acid molecule encoding human glucagon receptor and wherein thecompound specifically hybridizes with at least 90% complementaritywithin nucleotides 532 to 558 of a nucleic acid molecule having thesequence SEQ ID NO:4 and inhibits the expression of the human glucagonreceptor.
 27. The method of claim 26 wherein the human suffers fromdiabetes, obesity or insulin resistance.
 28. The method of claim 26wherein the blood glucose levels are plasma glucose levels.
 29. Themethod of claim 17, wherein the compound is at least 95% complementaryto SEQ ID NO:
 4. 30. The method of claim 17, wherein the compound is 15to 30 nucleobases in length.
 31. The method of claim 17, wherein thecompound is 20 nucleobases in length.
 32. The method of claim 17,wherein the compound comprises an oligonucleotide.
 33. The method ofclaim 32, wherein the oligonucleotide is chimeric.
 34. The method ofclaim 17, wherein the compound comprises at least one chemicalmodification.
 35. The method of claim 34, wherein the compound comprisesat least one modified internucleoside linkage, sugar moiety, ornucleobase.
 36. The method of claim 35, wherein the compound comprisesat least one 2′-O-(2-methoxyethyl) sugar moiety.
 37. The method of claim35, wherein the compound comprises at least one phosphorothioateinternucleoside linkage.
 38. The method of claim 35, wherein thecompound comprises at least one 5-methylcytosine.
 39. A method of claim33, wherein every internucleoside linkage of the oligonucleotide is aphosphorothioate linkage, nucleobases 1-5 and 16-20 of theoligonucleotide comprise a 2′-O-(2-methoxyethyl) modification and everycytidine residue comprises a 5-methyl modification.
 40. The method ofclaim 17, wherein the compound is a sodium salt.
 41. The method of claim17, wherein the compound is administered as a composition comprising apharmaceutically acceptable carrier, diluent or excipient.
 42. Themethod of claim 41, wherein the composition farther comprises acolloidal dispersion system.
 43. The method of claim 18, whereinadministering comprises delivering a plurality of doses.
 44. The methodof claim 43, wherein each dose of said plurality of doses comprises fromabout 1 mg about 20 mg of the compound per kg of body weight.
 45. Themethod of claim 17, wherein the compound is 100% complementary to SEQ IDNO:4.
 46. The method of claim 17, wherein the compound targets andspecifically hybridizes within nucleotides 532 to 558.